FSA Project Code: FS235003 (P01009) Cefas ref: C3027 FSA Project Title: Investigation into the prevalence, distribution and levels of norovirus titre in oyster harvesting areas in the UK Start Date: 15th September 2008 End Date: 31st August 2011 Project Leader's Name: Dr James Lowther Institution: CEFAS Weymouth Laboratory The Nothe Barrack Road Weymouth DT4 8UB Date of submission: 11th August 2011 Executive summary

In this two year study, monthly samples of Pacific (Crassostrea gigas) or native oysters (Ostrea edulis) from 39 production areas around the UK were tested for norovirus genogroups I (GI) and II (GII) using a quantitative real-time RT-PCR method. All samples were also tested for E. coli using the regulatory reference method. All sites sampled were designated and classified under EU Regulation 854/2004 and thus available for commercial production. Classifications ranged from class A to class C. Sites included in the study were selected using a risk matrix score to ensure a representative selection of likely faecal contamination levels.

Norovirus was detected in 76.2% (643/844 of samples), with similar prevalences in the two species of oysters tested (76.1% (468/615) for C. gigas and 76.4% (175/229) for O. edulis). There was a marked seasonality with a positivity rate of 90.0% (379/421) for samples taken between October and March compared with 62.4% (264/423) for samples taken between April and September.

Quantification of positive samples revealed that the majority (52.1%; 335/643) were below the limit of quantification (100 detectable genome copies/g digestive tissues) for both genogroups. However a number of samples (1.4% of all positives; 9/463) contained levels in excess of 10,000 copies/g. As with prevalence, average quantities varied markedly between seasons, with highest levels detected between December and March. The scores for norovirus GI and GII in individual samples were significantly correlated with each other, however results for norovirus GII tended on average to be higher than those for GI.

All 39 sites tested provided at least one norovirus positive result, although prevalence varied from 21% (5/24 samples) to 100% (20/20 samples). Norovirus levels varied markedly between sites with some sites scoring consistently over 1,000 copies/g during the winter while others rarely or never exceeded 100 copies/g.

This study also examined relationships between norovirus contamination and potential risk indicators. A statistically significant correlation between norovirus levels and harvesting area classification was observed. Furthermore a statistically significant and predictive correlation was found between E. coli and norovirus levels when data was analysed by site rather than by sample. This finding provides support for the use of E. coli as an indicator organism for classification purposes. Strong correlations between norovirus contamination and environmental temperatures were also found on both a nationwide average and in the majority of cases a site-by-site basis.

A statistically significant correlation was also observed between the site-specific risk scores initially assigned and the norovirus contamination levels observed in the study. We were able to further improve the predictive power of the risk matrix based on a combination of the E. coli contamination profile for the site and the seawater temperature at the time of sampling. This approach could assist risk management in relation to, for example, highlighting the need for virus monitoring or enhanced operator control measures.

In summary the data generated in this study provides a systematic analysis of norovirus contamination in classified oyster production areas in the UK. The data will help to inform FSA policy on the reduction of viral contamination of bivalve molluscs and to instruct a coherent EU negotiating position. Acknowledgements

This project was funded by the Food Standards Agency, UK. We would like to thank all participating oyster producers and local authority sampling officers for their co-operation and contributions, without which this project would not have been possible. In addition the Shellfish Association of Great Britain provided valuable assistance in recruiting participants.

## Contents

Executive summary ........................................... i\
Acknowledgements ........................................... ii\
Contents ....................................................... iii\
List of Figures ................................................ v\
List of Tables ................................................ vi\
1.0 Introduction .............................................. 1\
2.0 Aims and Objectives ..................................... 2\
3.0 Methods .................................................... 2\
3.1 Sample collection ..................................... 2\
3.2 Detection and quantification of GI and GII norovirus 2\
3.3 Detection and quantification of *Escherichia coli* 4\
3.4 Statistical analysis ................................... 5\
4.0 Comparison of native and Pacific oysters as a test matrix 5\
4.1 Introduction .......................................... 5\
4.2 Experimental approach ............................... 5\
4.3 Results ................................................. 6\
4.4 Conclusion ............................................. 6\
5.0 Selection of oyster sites for surveillance ............... 7\
5.1 Risk Assessment ....................................... 7\
5.2 Site selection ......................................... 8\
6.0 Surveillance results ..................................... 10\
6.1 Prevalence of norovirus in tested samples .......... 10\
6.2 Levels of norovirus in tested samples ............. 11\
6.3 Comparison of project year 1 and year 2 .......... 13 6.4 Comparison of different sites 6.5 Comparison of different regions 6.6 Comparison of different oyster species 6.7 Comparison of shellfish harvesting area classifications 6.8 Correlation of norovirus with *E. coli* 6.9 Correlation of norovirus in oysters with clinical laboratory reports 6.10 Correlation of norovirus in oysters with environmental temperature data 6.11 Correlation of norovirus in oysters with risk scores 6.12 Further development of a risk scoring matrix

7.0 Discussion

8.0 References

Appendices

Appendix 1 Oyster species comparison results Appendix 2 Surveillance sample results Appendix 3 Statistical analysis List of Figures

Figure 1: Recovery of norovirus genogroup II from aliquots of native (*O. edulis*) and Pacific (*C. gigas*) oysters contaminated with faecal suspension.

Figure 2: Month-by-month percentages of samples positive for norovirus.

Figure 3: Monthly geometric mean norovirus levels.

Figure 4: Monthly proportion of samples giving total norovirus results in different quantity brackets (copies/g).

Figure 5: Month-by-month results for representative low, medium and high norovirus sites.

Figure 6: Monthly geometric mean norovirus levels for different UK regions.

Figure 7: Within-site geometric means of individual sample norovirus scores grouped by UK region.

Figure 8: Within-site geometric means of individual sample norovirus scores grouped by species.

Figure 9: Within-site geometric means of individual sample norovirus scores grouped by classification.

Figure 10: Norovirus vs. *E. coli* for individual samples.

Figure 11: Norovirus vs. *E. coli*; comparison of within-site geometric means of individual sample scores (all months).

Figure 12: Norovirus vs. *E. coli*; comparison of within-site geometric means of individual sample scores (October-March).

Figure 13: Comparison of monthly geometric mean norovirus levels in oysters with lab reports received by the HPA.

Figure 14: Comparison of monthly geometric mean norovirus levels in oysters with national average temperatures.

Figure 15: Norovirus vs. seawater temperature; comparison of individual sample norovirus scores with seawater temperatures at the time of sampling. Figure 16: Norovirus vs. risk scores; comparison of within-site geometric means of individual sample norovirus scores with risk scores (England and Wales).

Figure 17: Norovirus vs. sampling risk scores.

Figure 18: Proportion of samples giving total norovirus results in different quantity brackets (copies/g) for different sampling risk scores.

List of Tables

Table 1: Risk based scoring system (risk matrix) 7 Table 2: Population cut-off for risk assessment of oyster sites in Scotland 8 Table 3: Sites included in surveillance 9 Table 4: Two-by-two contingency table of samples testing positive and not detected for norovirus GI and GII 10 Table 5: Two-by-two contingency table of positive and not detected samples taken during summer and winter 11 Table 6: Number of samples giving norovirus results in different quantity brackets 11 Table 7: Two-by-two contingency table of positive and not detected samples taken during project years 1 and 2 13 Table 8: Two-by-two contingency table of norovirus scores \<100 or >100 copies/g from samples with sampling risk scores \<3.0 or >3.0 23 1.0 Introduction

Contamination of bivalve shellfish with norovirus from human faecal sources is recognised as a major human health risk (Lees, 2000). Risk assessment and management currently relies on the use of *Escherichia coli* as an indicator of faecal (sewage) contamination in shellfish (Anon, 2004). However, this approach has been repeatedly demonstrated to inadequately contain the risk from human enteric viruses (Ang, 1998; Chalmers & McMillan, 1995; Gill *et al*., 1983). Over the last decade considerable progress has been made towards development of sensitive detection methods for norovirus in molluscan shellfish and there are numerous publications describing various approaches to virus testing (Höhne and Schreier, 2004; Jothikumar *et al*., 2005; Le Guyader *et al*., 2009; Loisy *et al*., 2005). All available methods are based on detection of virus genome using molecular techniques (PCR), as successful cultivation of norovirus by conventional virological techniques has been reported only rarely and is not feasible for application to foodstuffs. At present no internationally accepted reference method exists, however the European Committee on Normalisation (CEN) has an active working group, chaired by Cefas, addressing the development of a standard method for detection of norovirus and HAV in foodstuffs (including bivalve shellfish). A draft method is nearing completion (Lees and CEN WG6 TAG4, 2010) and this will be succeeded by an international method validation exercise. The timetable for publication of the CEN Technical Specification is 2012. EU legislative text foreshadows the adoption of virus controls when the methods are sufficiently developed and available for use (Anon, 2004; Anon, 2005). Thus it is possible that following publication of a CEN standard viral controls may be adopted into Community feed and food hygiene legislation. In preparation for possible legislative viral standards across the EU it has become essential to gain information about the practical application of the methods and potential impact on UK shellfisheries.

Previously published and informal studies examining norovirus in UK oysters have revealed the presence of viral RNA in a significant proportion of samples, as well as indicating a clear seasonal pattern of contamination with higher prevalence and levels detected during the winter months from October to March (Lowther *et al*., 2008; Lowther *et al*., 2010). These studies have however all targeted specific production areas due to for example historical links to outbreaks of gastroenteritis, and the sampling plans were developed on an *ad hoc* basis according to the particular needs of the project. This project aimed to address these issues through application of a systematic sampling plan to oysters collected from a representative selection of UK oyster production sites and by testing these oysters for norovirus genogroups I and II using the most up-to-date quantitative methods based on the developing CEN standard. In this way the study seeks to generate systematic quantitative data on the occurrence, prevalence and levels of norovirus in UK production areas. The outputs of this project may help to inform FSA discussions with respect to reducing viral contamination of bivalve molluscs. 2.0 Aims and objectives The aim of this project was to determine the rates of prevalence and typical levels of norovirus in oysters from a representative selection of sites from around the UK. The project also aimed to assess any seasonal variations and any relationships of norovirus levels with *E. coli* and other risk factors (such as population, environmental temperature, etc.).

3.0 Methods

3.1 Sample collection Samples were collected by the relevant local authority sampling officers in parallel to the collection of samples for the statutory classification monitoring programme. Sampling officers were asked to collect one sample per month of thirty oysters from each selected site from May 2009 to April 2011 inclusive. These were dispatched to the laboratory under temperature controlled conditions. Where practicable, water temperatures at the sampling site were taken using an approved method ([http://www.cefas.defra.gov.uk/media/436986/samplingprotocolforlocalauthorities%5b1%5d.pdf](http://www.cefas.defra.gov.uk/media/436986/samplingprotocolforlocalauthorities%5b1%5d.pdf)) at the time of sampling.

3.2 Detection and quantification of GI and GII norovirus

3.2.1 Preparation of oyster homogenate Ten oysters from each sample under test were cleaned, opened and removed from their shells. The peripheral flesh and organs of the animals were then cut away from the digestive gland and discarded. The glands of the oysters were pooled together then chopped finely with a razor-blade. A 2g portion of the chopped glands was added to a 50ml centrifuge tube for immediate testing and the remainder was retained frozen at -20°C. A 10µl volume of Mengo virus strain vMC0 (to act as a process control) and 2ml of 100 µg/ml proteinase K solution (30U/mg; Promega) were then added to the chopped glands in the 50ml tube. The tube was then incubated at 37°C with shaking at 320 rpm for a duration of 1 hour, and subsequently incubated at 60°C for a duration of 15 min. The tube was centrifuged at 3000 x g for 5 min, then the soluble portion (homogenate) poured into a clean tube. Finally the volume of the homogenate was measured using a pipette, then it was retained at 4°C for RNA purification and RT-PCR.

3.2.2 Purification of viral RNA Viral RNA extraction was carried out using NucliSens magnetic extraction technology (BioMerieux). For each sample a 500µl aliquot of sample homogenate was added to 2ml NucliSens lysis buffer in a 15ml centrifuge tube. In addition for each batch of samples tested negative control and positive extraction control samples consisting of 500µl water only and norovirus positive oyster homogenate (derived from bioaccumulated animals) respectively were also prepared and tested in parallel. Samples and controls were vortexed briefly then incubated at room temperature for 10 min before 50µl magnetic silica was added to each tube and the samples incubated at room temperature for a further 10 min. The tubes were centrifuged at 1500 x g for 2 min and the supernatants removed by aspiration. The pelleted silica beads were resuspended in 400µl wash buffer 1 then transferred to individually labelled 1.5ml tubes on the MiniMag extraction station. The magnet of the MiniMag was raised to collect the silica beads on the walls of the tubes, then the beads washed for 30 secs using the wash function of the MiniMag. The supernatants were removed by aspiration, then the magnet lowered and the silica beads resuspended with 400µl of wash buffer 1, washed and the supernatant aspirated as before. The resuspension/wash/aspiration cycle was then repeated using 500µl wash buffer 2 then 500µl wash buffer 3 (wash for 15 secs only). The pelleted silica beads were then resuspended with 100µl of elution buffer. The tubes were capped and transferred to the thermoshaker at 60°C and shaken at 1400 rpm for 5 min, to allow elution of DNA and RNA from the silica beads. After elution the tubes were transferred to a portable magnet to collect the silica beads on the walls of the tubes, then the supernatant (sample nucleic acid (NA) extract) was transferred to a clean 0.5ml tube and stored at -20°C until required for reverse transcription.

3.2.3 One-step RT-PCR For GI QNIF4 (da Silva et al., 2007), and NV1LCR primers (Svraka et al., 2007), and TM9 probe (Höhne and Schreier, 2006) were used. For GII, QNIF2 (Loisy et al., 2005), and COG2R primers (Kageyama et al., 2003), and QNIFS probe (Loisy et al., 2005) were used. For each sample or extraction control and both norovirus genogroups three aliquots of 5µl NA extract were added to adjacent wells of a 96-well optical reaction plate and made up to 25µl with (GI or GII) TaqMan reaction mix (final concentration of 1x each Ultrasense reaction mix, Rox reference dye and RNA Ultrasense enzyme mix (Invitrogen), 500 nM forward primer, 900 nM reverse primer, and 250 nM probe). Positive (dilution series prepared from a known concentration of plasmid carrying a copy of the target sequence) and negative (three aliquots of 5µl water only) PCR control materials were also tested. The plate was placed in a Stratagene Mx3005P real-time PCR machine with the following amplification program; 55°C for 60 min, then 95°C for 5 min, followed by 45 cycles of 95°C for 15 secs, 60°C for 1 min and 65°C for 1 min. For analysis, threshold values were set at 0.20 fluorescence units, then threshold cycle (Ct) values were determined using the Mx3005P system software. Unexpected results in any positive or negative extraction or RT-PCR control triggered retesting of any affected samples.

3.2.4 Quantification of norovirus using dsDNA standard curve analysis Norovirus titres were calculated based upon the principles of the forthcoming CEN standard method for quantification of viruses from CEN/TC275/WG6/TAG4. On each TaqMan run a log dilution series of dsDNA control corresponding to a range of approx 1 to 10,000 template copies/µl (quantified using spectrophotometry at 260nm) was included. The Ct values from this dilution series were then used to produce a standard curve. For each TaqMan replicate for the samples under test a quantity in copies/µl was determined using the corresponding standard curve. Not detected replicates were ascribed a quantity of zero. The average quantities from the three replicates in each norovirus genogroup-specific TaqMan assay were calculated to give an overall quantity in detectable copies/µl NA extract for that sample and genogroup. This was multiplied by 100 to give detectable copies/test portion of homogenate (500µl), further multiplied by a factor based on the proportion of the complete homogenate tested, and finally divided by 2 to give a quantity in detectable genome copies/g digestive gland. This value does not take account of any losses during processing or RT-PCR inhibition and should be regarded as a minimum. Uncertainty of measurement of this method has been estimated at ±0.17 log\_{10} copies/g. 3.2.5 Calculation of RT-PCR efficiency For each sample and each norovirus genogroup one aliquot of 5μl NA extract was added to a 96-well optical reaction plate and made up to 25μl with (GI or GII) TaqMan reaction mix (final concentrations as described above). A 1μl volume containing a high concentration of GI or GII RNA sequences (produced by in vitro transcription from the dsDNA control) was then added to each aliquot of sample NA extract in addition to a 5μl aliquot of water in a separate well. The plate was placed in a Stratagene Mx3005P real-time PCR machine and amplified using the program described above. The percentage RT-PCR efficiency for each sample and each genogroup was determined by comparing the Ct values for the sample NA extract plus control NA extract with that for the water plus control NA extract. Any sample with an RT-PCR efficiency of \<25% was subjected to retesting, in the first instance by re-extracting the viral RNA from stored homogenate, then by testing stored digestive glands. Results for any sample providing three RT-PCR efficiency results of \<25% were considered invalid and are recorded as “no result” in Appendix 3.

3.2.6 Calculation of extraction efficiency For each sample two aliquots of 5μl NA extract were added to adjacent wells of a 96-well optical reaction plate and made up to 25μl with Mengo virus-specific TaqMan reaction mix (final concentrations as described above). A dilution series prepared from the Mengo virus process control material was also tested. The plate was placed in a Stratagene Mx3005P real-time PCR machine and amplified using the program described above. The percentage extraction efficiency for each sample was determined by comparing the Ct values for the sample NA extract with those for the Mengo virus dilution series. Any sample with an extraction efficiency of \<1% was subjected to retesting, in the first instance by re-extracting the viral RNA from stored homogenate, then by testing stored digestive glands. Results for any sample providing three extraction efficiency results of \<1% were considered invalid and are recorded in Appendix 3 as “no result”.

3.2.7 Expression of results Samples where all three RT-PCR reactions for a given genogroup were negative are described as “not detected; \<40 copies/g” in Appendix 3 (40 copies/g is the practical limit of detection of the method). Samples where the calculated quantity (as described above) was >500 copies/g are described as “positive; x copies/g” where x is the calculated quantity. Samples where the calculated quantity was between 100 and 500 copies/g are described as “positive; x copies/g” unless there were significant differences between results in individual RT-PCR reactions, in which case they are described as “positive; 100-500 copies/g”. Samples where the calculated quantity was \<100 copies/g (100 copies/g is the practical limit of quantification of the method) are described as “positive; \<100 copies/g”.

3.3 Detection and quantification of Escherichia coli

3.3.1 Preparation of oyster homogenate Ten oysters from each sample under test were cleaned, opened and removed from their shells. The flesh was added to a blender with 2 volumes (per weight) of a 0.1% (w/v) solution of peptone (Oxoid) then homogenized using 4 bursts of 15 seconds at high speed. The homogenate was then retained at 4ºC for up to 24 hrs prior to enumeration. 3.3.2 Enumeration of *E. coli*

Oyster homogenates were assayed for *E. coli* using a standard most probable number method derived from ISO TS 16649-3. The homogenate was initially diluted using 0.1% (w/v) peptone to a concentration of 10% (v/v) (“neat”). A serial log$\_{10}$ dilution series from neat to $10^{-2}$ was then prepared. Diluted homogenate was inoculated into five tubes containing mineral-modified glutamate broth (Oxoid) for each of the three dilutions and the tubes were then incubated at 37°C for 18-24 hrs. Confirmation of acid producing tubes was carried out by subculture onto a chromogenic agar containing 5-bromo-4-chloro-3-indolyl-β-D glucoronide (LabM) and subsequent incubation at 44°C for at further 18-24 hrs. Blue-green colonies indicative of the action of β-glucoronidase activity were considered to be *E. coli* positive. The most probable number (MPN) was derived by reference to MPN tables contained within ISO 7218. The theoretical limit of detection of the assay was 20 MPN/100g shellfish and intravalvular fluid.

3.4 Statistical analysis

All statistical analysis was carried out using Minitab 16 software. For the purposes of analysis samples giving not detected results for a particular norovirus genogroup were scored 20 copies/g for that genogroup (half the limit of detection (LOD) of 40 copies/g). Samples giving positive results below the limit of quantification (LOQ; 100 copies/g) were scored 50 copies/g. For the purposes of figures and analyses where total counts of norovirus (GI and GII summed) were used, samples giving positive results \<LOQ for both genogroup scored 100 copies/g; such results were included in the \<100 copies/g bracket where such brackets were used. For *E. coli* samples giving results \<20 MPN/100g were scored 10 MPN/100g.

4.0 Comparison of native and Pacific oysters as a test matrix

4.1 Introduction

Prior to this project the laboratory had previous experience of testing large numbers of samples of Pacific oysters (*Crassostrea gigas*) for norovirus using the detection methods described but had tested comparatively small numbers of samples of native oysters (*Ostrea edulis*). For this reason, prior to selection of sites for surveillance it was necessary to determine the suitability of the test method for use with both species of oysters.

4.2 Experimental approach

For both oyster species, a number of animals were opened and the digestive glands excised as described in section 3.1.1. Separately for each species, glands were pooled together until >10g was present, chopped finely with a razor-blade, and apportioned in 2g aliquots into five tubes. Each aliquot was artificially contaminated with an identical volume of norovirus GII positive faecal suspension, then subjected to Proteinase K treatment, RNA purification in duplicate and quantitative RT-PCR for GII as described in section 3.1.

This procedure was repeated a total of three times using different contamination levels designed to result in approximate concentrations of 500,000, 50,000 and 5,000 copies/g respectively (‘high’, ‘medium’ and ‘low’). 4.3 Results Quantities in copies/g were calculated for all extractions (see Appendix 1). Results for duplicate extractions from the same homogenate were then averaged (Figure 1). Two-way analysis of variance of the log\_{10} transformed averaged data revealed that as expected contamination level had a significant impact on calculated quantities (p\<0.001), however there was no significant difference between species (p=0.232), and there was also no significant interaction between species and contamination level (p=0.233; see Appendix 3 for statistical analysis).

4.4 Conclusion Based on the data generated there was no evidence that the performance of the selected detection method varies significantly between the two oyster species of commercial significance in the UK. Both species were therefore included in the surveillance part of this project. 5.0 Selection of oyster sites for surveillance

5.1 Risk Assessment

5.1.1 Introduction In this study risk scores were used to rank sites in order to provide a more scientific basis for sampling site selection. They should not be over-interpreted since the actual scores are susceptible to the weightings applied for each factor and for many risks the correct public health weighting cannot readily be established. In addition risk scores derived for England and Wales, and for Scotland, cannot be directly compared since it was necessary to use different population geographic factors (see below). Risk assessment was not carried out for sites in Northern Ireland due to the small number of potential candidate sites.

5.1.2 England and Wales All active representative monitoring points (RMPs) with 75% or more sampling compliance in official control monitoring in the previous sampling year were selected for risk assessment (total 121 sites from 38 production areas). Each site was assigned a risk score based on a risk matrix as detailed in Table 1.

Table 1: Risk based scoring system (risk matrix)

| Factor | Low (score 1) | Medium (score 3) | High (score 5) | |--------|---------------|------------------|---------------| | 1 | Classification status 2006 | A or unclassified | A/B, B, B-LT or provisional classification | A/B/C, B/C or C | | 2 | Classification status 2007 | As above | As above | As above | | 3 | Classification status 2008 | As above | As above | As above | | 4 | Changes in classification status | None (or change from not classified or provisional) | One in three years | Two in three years | | 5 | Unusual *E. coli* results (per 100g) 2006 | All results normal relative to annual classification scores 1 | Each “high” result relative to annual classification scores 3 | Each “very high” result relative to annual classification scores 5 | | | Class A ≤230 | Class B ≤4,600 | Class C ≤10,000 | Class A 230-1,000 | Class B 4,600-18,000 | Class C 10,000-18,000 | Class A >1,000 | Class B >18,000 | Class C >18,000 | | 6 | Unusual *E. coli* results (per 100g) 2007 | As above | As above | As above | | 7 | Unusual *E. coli* results (per 100g) 2008 | As above | As above | As above | | 8 | Population in adjacent area | \<200,000 | 200,000-399,999 | ≥400,000 | | 9 | Confirmed outbreaks | None | - | One or more in three years | | 10 | Unconfirmed outbreaks | None | Between one and three in three years | More than three in three years |

- Split classifications considered at an equivalent annual classification, e.g. results for an A/B area judged against class B criteria.
- Where no results were taken site scored 3 for the year in question.
- Working range TS ISO16649-3 is generally up to 18,000 *E. coli* MPN/100g so all results above this are reported as >18,000.
- Including all census areas of which at least part lies within 10km of the sampling point.
- Reported by Health Protection Agency or Health Protection Scotland for the years 2005, 2006 and 2007.
- Notified to FSA or the National Reference Laboratory for the years 2005, 2006 and 2007. 5.1.3 Scotland Generation of risk data was carried out as part of FSAS SPIN 069 as previously described (Price-Hayward, 2010). All active production areas were selected for risk assessment (total 41 areas). Each area was assigned a risk score based on a risk matrix as detailed in Table 1, with the exception of different population cut-offs (Table 2) to take account of differences in population pressures.

Table 2: Population cut-off for risk assessment of oyster sites in Scotland

| Factor | Low (score 1) | Medium (score 3) | High (score 5) | |-------------------------------|---------------|------------------|----------------| | Population in adjacent area | \<600 | 600-1,200 | ≥1,200 |

\*Including all census areas of which at least part lies within 2km of the sampling point.

5.2 Site selection

5.2.1 England and Wales A total of 31 sites (20 Pacific and 11 native oysters, Table 3) were selected using the following criteria.

1. sites with a broad range of risk scores
2. sites from a broad range of geographical locations
3. sites from a wide range of production areas; oyster production in England and Wales is concentrated on the South and East coasts so these regions are well represented

5.2.2 Northern Ireland Two sites (both Pacific oysters) were selected in direct consultation with FSA Northern Ireland.

5.2.3 Scotland A total of 6 sites (all Pacific oysters) were selected using the same criteria as outlined above. Table 3: Sites included in surveillance

| Site number | Region | Species | Risk score | Classification | |-------------|---------------------------------------------|---------|------------|----------------| | 1 | England - East Coast | C. gigas| 24 | B | | 2 | England - East Coast | C. gigas| 23 | B | | 3 | England - East Coast | O. edulis| 16 | B | | 4 | England - East Coast | C. gigas| 18 | B | | 5 | England - East Coast | C. gigas| 25 | B | | 6 | England - East Coast | O. edulis| 20 | B | | 7 | England - East Coast | C. gigas| 22 | B | | 8 | England - East Coast | C. gigas| 18 | B | | 9 | England - East Coast | O. edulis| 22 | B | | 10 | England - East Coast | O. edulis| 12 | A | | 11 | England - East Coast | C. gigas| 30 | C | | 12 | England - East Coast | C. gigas| 20 | B | | 13 | England - South Coast | C. gigas| 38 | B | | 14 | England - South Coast | O. edulis| 25 | C | | 15 | England - South Coast | C. gigas| 22 | B | | 16 | England - South Coast | O. edulis| 20 | B | | 17 | England - South Coast | O. edulis| 27 | B | | 18 | England - South Coast | C. gigas| 17 | B | | 19 | England - South Coast | C. gigas| 14 | B | | 20 | England - South Coast | C. gigas| 22 | B | | 21 | England - South Coast | C. gigas| 14 | B | | 22 | England - South Coast | C. gigas| 20 | B | | 23 | England - South Coast | O. edulis| 22 | B | | 24 | England - South Coast | O. edulis| 32 | B | | 25 | England - South Coast | O. edulis| 54 | B | | 26 | England - West Coast, Wales & NI | C. gigas| 12 | B | | 27 | England - West Coast, Wales & NI | C. gigas| 14 | B | | 28 | England - West Coast, Wales & NI | C. gigas| 20 | B | | 29 | England - West Coast, Wales & NI | C. gigas| n/a | B | | 30 | England - West Coast, Wales & NI | C. gigas| 21 | B | | 31 | England - West Coast, Wales & NI | C. gigas| n/a | A | | 32 | England - West Coast, Wales & NI | O. edulis| 20 | B | | 33 | England - West Coast, Wales & NI | C. gigas| 18 | B | | 34 | Scotland | C. gigas| 32 | A | | 35 | Scotland | C. gigas| 36 | B | | 36 | Scotland | C. gigas| 16 | A | | 37 | Scotland | C. gigas| 50 | B | | 38 | Scotland | C. gigas| 20 | A | | 39 | Scotland | C. gigas| 26 | A |

1 For sites with seasonal classification, or where classification changed during the sampling period, the classification covering the majority of samples taken is shown. 2 Coast of England from Eastern border with Scotland to North Foreland. 3 Coast of England from North Foreland to Land’s End. 4 Coast of England and Wales from Land’s End to Western border with Scotland, plus coast of Northern Ireland. 5 Two sites without risk scores included to provide appropriate geographical coverage. 6.0 Surveillance results

6.1 Prevalence of norovirus in tested samples

Full results for each site are included in Appendix 2. Of 857 samples tested 844 (98.5%) provided valid results (i.e. with acceptable extraction and amplification efficiencies) for norovirus GI and GII. Of these 844, 643 samples (76.2% [95% CI; 73.1-79.0%]) tested positive for norovirus; 402 (47.6% [95% CI; 44.2-51.1%]) were positive for both genogroups, 176 (20.9% [95% CI; 18.2-23.8%]) were positive for GI only and 65 (7.7% [95% CI; 6.0-9.8%]) were positive for GII only. Analysis of the two-by-two contingency table of positive and not detected results for GI and GII (Table 4) using Fisher’s exact test showed a highly significant difference between prevalence for the two genogroups (13.2% more GI positives; p\<0.0001). Norovirus prevalence varied from month-to-month (Figure 2) ranging from a maximum of 100% positive (38/38 samples; 95% CI; 88.6-100%) in February 2010 to a minimum of 45.7% samples positive (16/35; 95% CI; 29.2-63.1%) in September 2010. Analysis of the two-by-two contingency table of positive and not detected samples from the summer months (April-September) and the winter months (October-March; Table 5) using Fisher’s exact test showed a highly significant difference between prevalence in the two seasons (27.6% higher in the winter; p\<0.0001).

![Figure 2: Month-by-month percentages of samples positive for norovirus.](image)

Table 4: Two-by-two contingency table of samples testing positive and not detected for norovirus GI and GII

| Result | Positive | Not detected | |--------|----------|--------------| | Norovirus GI | 578 (68.5%) [95% CI; 65.2-71.6%] | 266 (31.5%) [95% CI; 28.4-34.8%] | | Norovirus GII | 467 (55.3%) [95% CI; 51.9-58.7%] | 377 (44.7%) [95% CI; 41.3-48.1%] |

*Percentage of samples testing positive or not detected in period shown in brackets* Table 5: Two-by-two contingency table of positive and not detected samples taken during summer and winter

| Norovirus result | Positive | Not detected | |------------------|----------|--------------| | Summer (April-September) | 264 (62.4%) | 159 (37.6%) | | [95% CI; 57.6-67.0%] | [95% CI; 33.0-42.4%] | | Winter (October-March) | 379 (90.0%) | 42 (10.0%) | | [95% CI; 86.7-92.6%] | [95% CI; 7.4-13.3%] |

Percentage of samples testing positive or not detected in period shown in brackets

6.2 Levels of norovirus in tested samples

Levels of norovirus in tested samples varied widely. For norovirus GI the maximum recorded quantity was 16,507 copies/g and for GII it was 18,024 copies/g. However a large proportion of positive results (65.1% for GI [376/578 samples; 95% CI; 61-68.9%] and 42.8% for GII [200/467; 95% CI; 38.3-47.5%]) were below the limit of quantification of the method (100 copies/g). The numbers and percentages of samples giving GI, GII and total norovirus results in different quantity brackets is shown in Table 6.

Table 6: Number of samples giving norovirus results in different quantity brackets

| Number of samples in norovirus quantity brackets (copies/g) | Not detected | \<100 copies/g | 100-999 copies/g | 1,000-10,000 copies/g | >10,000 copies/g | |-----------------------------------------------------------|--------------|---------------|------------------|----------------------|-----------------| | GI | 266 (31.5%) | 376 (44.5%) | 168 (19.9%) | 31 (3.7%) | 3 (0.4%) | | [95% CI; 28.4-34.8%] | [95% CI; 41.2-48.0%] | [95% CI; 17.3-22.8%] | [95% CI; 2.6-5.2%] | [95% CI; 0.1-1.1%] | | GII | 377 (44.7%) | 200 (23.7%) | 172 (20.4%) | 89 (10.5%) | 6 (0.7%) | | [95% CI; 41.3-48.1%] | [95% CI; 20.9-26.8%] | [95% CI; 17.7-23.3%] | [95% CI; 8.6-12.9%] | [95% CI; 0.3-1.6%] | | Sum | 201 (23.8%) | 335 (39.7%) | 185 (21.9%) | 114 (13.5%) | 9 (1.1%) | | [95% CI; 21.0-26.9%] | [95% CI; 36.4-43.1%] | [95% CI; 19.2-24.9%] | [95% CI; 11.3-16.1%] | [95% CI; 0.5-2.1%] |

Percentage of samples in given bracket shown in parentheses

As with prevalence (see section 6.1), norovirus levels varied seasonally (Figure 3) with annual peaks occurring between December and March and lowest levels recorded between May and August. Levels of norovirus GII were on average higher than those for GI. Analysis of GI and GII levels for the same samples (samples testing positive for one or both genogroups only) using the Wilcoxon signed ranks test indicated that within samples GII levels were significantly higher than GI (p=0.000). In order to confirm that this result was not unduly affected by the arbitrary scores given to \<LOD and \<LOQ results, sensitivity analysis was carried out by repeating the Wilcoxon test after rescoring \<LOD and \<LOQ results as 40 and 100 copies/g respectively as well as after rescoring \<LOD and \<LOQ results as 0 and 1 copy/g respectively. In both cases differences between the two genogroups were still found to be significant (p=0.000). The Spearman’s rank correlation coefficient between norovirus GI and GII scores for individual samples (r=0.622) indicated that these two variables were significantly correlated (p=0.000). Figure 3: Monthly geometric mean norovirus levels.

For each month of the project, the percentage of samples with total norovirus (GI plus GII summed) in different quantity brackets is shown in Figure 4. This figure illustrates the potential burden of different theoretical safety cut-offs for total norovirus on UK production areas at different times of year.

Figure 4: Monthly proportion of samples giving total norovirus results in different quantity brackets (copies/g). 6.3 Comparison of project year 1 and year 2

Overall prevalence, average levels and seasonal trends were broadly similar between the two years of the project (May 2009-April 2010 and May 2010-April 2011; Figures 2, 3 and 4). A greater proportion of samples tested positive in year 1 (Table 7), however this difference was not significant when analysed using Fisher’s exact test (p=0.090). On the other hand average levels were higher in year 2 (geomean 175 copies/g (95% CI; 151-203 copies/g) for year 2 compared with 144 copies/g (95% CI; 126-164 copies/g) for year 1. Analysis of levels from year 1 and 2 using the Kruskal-Wallis test showed no significant difference between the genogroups however (p=0.513).

Table 7: Two-by-two contingency table of positive and not detected samples taken during project years 1 and 2

| Norovirus result¹ | Positive | Not detected | |-------------------|----------|--------------| | Year 1 (May 2009-April 2010) | 337 (78.7%) [95% CI; 74.5-82.5%] | 91 (21.3%) [95% CI; 17.5-25.5%] | | Year 2 (May 2010-April 2011) | 306 (73.6%) [95% CI; 69-77.7%] | 110 (26.4%) [95% CI; 22.3-31%] |

¹Percentage of samples testing positive or not detected in period shown in brackets

6.4 Comparison of different sites

All 39 sites sampled during the project returned at least one positive result. Site-by-site prevalence varied from a minimum of 20.8% norovirus positive (5/24 samples [95% CI; 7.9-42.7%]) up to 100% positive (20/20 samples [95% CI; 80-100%]). Average norovirus levels varied very widely between sites, with the within-site geometric mean norovirus scores (GI and GII summed, samples not detected for both genogroups score 40) ranging from 50 (95% CI; 40-68 copies/g) – 2243 copies/g (95% CI; 883-5701 copies/g). To illustrate typical contamination patterns monthly results for three sites with comparatively low norovirus, three with medium norovirus and three with high norovirus are shown in Figure 5. Figure 5: Month-by-month results for representative low, medium and high norovirus sites. Low, medium and high norovirus sites shown in green, yellow and red tones respectively.

6.5 Comparison of different regions

The winter seasonality in the data set as a whole was also apparent when the different regions of the UK were examined separately, although average contamination levels varied (Figure 6). Average levels for sites in Scotland in particular seemed lower than for other regions. Within-site geometric mean norovirus scores grouped by region are shown in Figure 7. Median values for these sets ranged from 82 copies/g (Scotland [95% CI; 50-169 copies/g]) to 226 copies/g (England – East Coast [95% CI; 73-537 copies/g]) and comparison of the sets using the Kruskal-Wallis test revealed a significant difference in average norovirus contamination for tested sites for the four regions of the UK (p=0.029). Post hoc analysis using pairwise comparisons indicated that there were significant differences in average norovirus contamination between tested sites in Scotland and both England – East Coast and England – South Coast, but no differences between any of the other pairs.

Figure 6: Monthly geometric mean norovirus levels for different UK regions. 6.6 Comparison of different oyster species

The 39 sites analysed included 28 for Pacific oysters (C. gigas) and 11 for native oysters (O. edulis). The prevalence of norovirus in the two species was highly similar with 468 out of 615 C. gigas and 175 out of 229 O. edulis samples testing positive (76.1% [95% CI; 72.5-79.4%] and 76.4% [95% CI; 70.3-81.7%] respectively). Within-site geometric mean norovirus scores grouped by species are shown in Figure 8. Comparison of the two sets using the Kruskal-Wallis test revealed no significant difference in median norovirus contamination for tested sites for the two species (p=0.901; C. gigas median = 165 copies/g [95% CI; 102-197 copies/g], O. edulis median = 174 copies/g [95% CI; 59-328 copies/g]). 6.7 Comparison of shellfish harvesting area classifications

The 39 sites analysed included 6 sites classified A for the majority of the sampling period, 31 sites classified B and 2 sites classified C. Within-site geometric mean norovirus scores grouped by classification are shown in Figure 9. Median values for these three sets increased with classification (82 copies/g [95% CI; 52-175 copies/g], 180 copies/g [95% CI; 116-209 copies/g] and 1,208 copies/g [range 174-2243 copies/g; too few measurements to calculate CI] for class A, B and C respectively) and comparison of the three sets using the Kruskal-Wallis test revealed a significant difference in average norovirus contamination for tested sites for the three classifications (p=0.018). Post hoc analysis using pairwise comparisons indicated that there were significant differences in average norovirus contamination between A and B and A and C classification sites, but not between B and C classification sites (although the analysis was confounded by the small number of C classification sites in the study).

![Figure 9](image)

**Figure 9:** Within-site geometric means of individual sample norovirus scores grouped by classification.

6.8 Correlation of norovirus with *E. coli*

All 857 samples received were tested for *E. coli* (see section 3.3.2). One sample produced an invalid result (non-interpretable MPN tube combination on successive tests) however valid results were obtained for all other samples (n=856). In total 514 out of 856 samples (60.0% [95% CI; 56.7-63.3%]) contained levels which would be compliant with the end product standard (≤230 MPN/100g); the highest level recorded was 16,000 MPN/100g (found in 3 samples). Norovirus scores for individual samples plotted against the corresponding *E. coli* result are shown in Figure 10. As can be seen from Figure 10 a large number of samples returned low levels for norovirus and high levels for *E. coli*, or vice versa with little visual evidence of any correlation. Supporting this, the Spearman’s rank correlation coefficient between norovirus and *E. coli* scores on an individual sample basis was relatively low (r=0.195). However the existence of a significant interdependence of the two variables was strongly supported statistically (p=0.000). The rate of norovirus positivity for samples ≤230 MPN/100g was 73.9% (376/509 samples giving valid norovirus results; 95% CI; 69.8-77.6%); above this level it was 79.6% (266/334; 95% CI; 74.8-83.7%).

Figure 10: Norovirus vs. *E. coli* for individual samples.

Figure 11: Norovirus vs. *E. coli*; comparison of within-site geometric means of individual sample scores (all months). Within-site geometric mean values for norovirus and *E. coli* are shown in Figure 11. The Spearman’s rank correlation coefficient was higher than for individual sample results (r=0.453), and indicated that these two variables were significantly correlated (p=0.004). When results from the winter months (October-March) were considered the strength of the relationship between norovirus and *E. coli* geometric mean values increased markedly (r = 0.676, p=0.000; Figure 12).

![Figure 12: Norovirus vs. E. coli; comparison of within-site geometric means of individual sample scores (October-March).](image)

### 6.9 Correlation of norovirus in oysters with clinical laboratory reports

Monthly geometric mean norovirus levels (both genogroups summed) for the samples tested in this study are shown in Figure 13 alongside monthly total numbers of laboratory reports of norovirus infections received by the Health Protection Agency from England and Wales and reported on their website, see e.g. [http://www.hpa.org.uk/Topics/InfectiousDiseases/InfectionsAZ/Norovirus](http://www.hpa.org.uk/Topics/InfectiousDiseases/InfectionsAZ/Norovirus)

For the period of the study the two variables shared a common winter seasonality and the Spearman’s rank correlation coefficient between them (r=0.761) indicated that they were significantly correlated (p=0.000). However in contrast to the peak for norovirus in oyster samples, more lab reports were received by the HPA in the winter of 2009/10 than in the following winter (2010/11) although lab reports tend to be related to outbreaks associated with healthcare institutions and may not reflect the overall levels of norovirus in the community. Figure 13: Comparison of monthly geometric mean norovirus levels in oysters with lab reports received by the HPA.

6.10 Correlation of norovirus in oysters with environmental temperature

Monthly geometric mean norovirus levels (both genogroups summed) for the samples tested in this study are shown in Figure 14 alongside monthly UK national average air temperatures (plotted with a reversed y axis to aid visualisation) as reported by the Met Office e.g. http://www.metoffice.gov.uk/climate/uk/actualmonthly

Figure 14: Comparison of monthly geometric mean norovirus levels in oysters with national average air temperatures. For the period of the study the two variables shared a very strong, inverse, common seasonality (highest norovirus during periods of lowest air temperature) and the Spearman’s rank correlation coefficient between them ($r=-0.877$) indicated that they were significantly dependent ($p=0.000$). The maximum monthly geometric mean for norovirus in oysters (January 2011) followed shortly after the lowest monthly average air temperature of $-1^\\circ C$ in December 2010.

Seawater temperatures at the time of sampling were available for all or most samples for 36 out of 39 sites (for some sites seawater temperatures for a proportion of samples were missing due to particular issues, tides, problems with equipment etc. at the time of sampling). In order to investigate whether there was a common (although possibly variable) within-site correlation between temperature and norovirus Spearman’s rank analysis was carried out on data from each site. In 27 out of 36 sites there was a significant negative correlation between water temperature and norovirus levels (correlation coefficients of $r=-0.460$ to $r=-0.879$, see Appendix 3). For illustrative purposes Figure 15 shows norovirus scores plotted against seawater temperatures for samples from two sites where there was a particularly strong relationship. Of those sites where no statistically significant correlation was apparent the majority had low overall norovirus contamination levels (5 out of 9 with a within-site geometric mean norovirus score less than 100 copies/g).

![Figure 15: Norovirus vs. seawater temperature; comparison of individual sample norovirus scores with seawater temperatures at the time of sampling.](image)

6.11 Correlation of norovirus in oysters with risk scores

Figure 16 shows within-site geometric mean norovirus scores for sites in England and Wales plotted against the risk scores for these sites (as calculated in section 5.1.1 and detailed in Table 3; risk scores for sites in Scotland used a different matrix and are therefore not included in this analysis). The Spearman’s rank correlation coefficient between the two variables ($r=0.546$) indicated that they were significantly correlated ($p=0.001$), illustrating the value of the risk-based site selection method used. 6.12 Further development of a risk scoring matrix

The elaboration of a predictive matrix for norovirus risk could be a useful risk management tool. A correlation was found between the risk scoring matrix developed for site selection and norovirus geometric mean scores (see Figure 16). Further analysis attempted to improve the predictive potential of this approach using the data obtained in this study to develop the model. The existence of a significant within-site relationship between *E. coli* and norovirus scores, particularly during the winter, has been shown in section 6.8 (see Figure 12). In addition a correlation between seawater temperatures and norovirus scores for the majority of sites was found (section 6.10), while the seasonal variation in norovirus risk was clearly apparent (sections 6.1 and 6.2). We therefore elaborated an improved risk scoring system based on these factors as follows:

Factor $a =$ within site geometric mean of *E. coli* scores from samples taken October-March (proxy for the overall norovirus risk of the site).

Factor $b = 20 - \\text{(seawater temperature at the time of sampling)}$; a minimum baseline value of 1 was used for this factor such that seawater temperatures $\\geq 19^\\circ C$ all score 1.

Factor $c =$ seasonal multiplier dependent on month of sampling as follows:

- May-August $= 0.75$
- March, April, September, October $= 1.00$
- November-February $= 1.25$

Risk score $= (\\log\_{10}(\\text{product of factors } a \\text{ and } b))$ multiplied by factor $c$.

![Figure 16: Norovirus vs. risk scores; comparison of within-site geometric means of individual sample norovirus scores with risk scores (England and Wales).](image) Following this approach risk scores were calculated for all samples where seawater temperature data existed. These are plotted against norovirus scores in Figure 17. The Spearman’s rank correlation coefficient between the two variables ($r=0.645$) indicated that they were significantly correlated ($p=0.000$).

![Figure 17: Norovirus vs. sampling risk scores.](image)

The distribution of norovirus results in different quantity brackets according to sampling risk score is shown in Figure 18. Defining a norovirus score of >100 copies/g as a gold standard positive and a sampling risk score of >3.0 as a test outcome positive, this risk scoring system applied to the data in this study would have had an overall accuracy of 76.1% (95% CI; 72.7-79.2%), a sensitivity of 88.8% (95% CI; 84.1-92.2%), a specificity of 68.7% (95% CI; 64.1-72.9%), a positive predictive value of 62.2% (95% CI; 57.0-67.2%) and a negative predictive value of 91.3% (95% CI; 87.6-94.0%) (see Table 8).

![Figure 18: Proportion of samples giving total norovirus results in different quantity brackets (copies/g) for different sampling risk scores.](image) Table 8: Two-by-two contingency table of norovirus scores \<100 or >100 copies/g from samples with sampling risk scores \<3.0 or >3.0

| Norovirus result | \<100 copies/g | >100 copies/g | |------------------|---------------|---------------| | Sampling risk score | \<3.0 | 305 | 29 | | | >3.0 | 139 | 229 |

7.0 Discussion

This study comprises the first systematic survey of the occurrence of norovirus in oyster production areas in the UK. The overall prevalence of samples testing positive for one or both norovirus genogroups was 76.2% (643/844). This was very similar for both oyster species tested in the study. GI norovirus was detected more frequently than GII (in 68.5% (578/844) and 55.3% (467/844) of samples respectively). This is in contrast to clinical samples where the overwhelming majority of norovirus outbreaks are caused by GII strains (Kroneman et al., 2008). However a greater frequency of norovirus GI in foodborne infections has previously been reported (Verhoef et al., 2010). Although relatively high prevalences (20-59%) have been recorded in several previous studies targeting oysters and other bivalve spp. (Costantini et al., 2006, Elamri et al., 2006, Hansman et al., 2008, Loisy et al., 2005, Lowther et al., 2008, Lowther et al., 2010) to the best of our knowledge the overall prevalence reported here is higher than that recorded in any other similar study. This may be due, at least in part, to progressive improvements in the sensitivity of the test method over recent years. It is also possible that the UK coastal environment is under greater population pressure and hence subject to more severe faecal contamination than other comparable countries. It is notable that within the different UK regions, norovirus levels appeared lowest in Scotland, where population pressures would be expected to be lowest.

The significant proportion of positive samples highlights the importance of quantification for assessing the burden of norovirus contamination. Of samples testing positive, in the majority of cases (52.1%; 335/643) neither genogroup contained levels in excess of the limit of quantitation (LOQ) of the assay (100 copies/g). On the other hand for a small number of samples results of >10,000 copies/g were recorded. However, it should be noted that it is not currently possible to determine the absolute human health risks posed by different levels of norovirus. There is growing evidence of a dose response for norovirus in human volunteer studies (Teunis et al., 2008) and also evidence from a restaurant oyster consumption study of a correlation between norovirus levels in the batch consumed and likelihood of illness (Lowther et al., 2010). Several agencies (e.g. the European Food Safety Authority) are currently considering possible norovirus threshold levels for risk management. It was noted that different possible threshold levels have widely different impacts. For example a threshold level (GI and GII combined) of 100 copies/g would have resulted in a sample failure rate of >60% during the winter (October to March) for both years of the study. In contrast a threshold level of 1,000 copies/g would have resulted in a sample failure rate of \<30%.

The data presented in this study should prove valuable in determining the impacts of the use of such threshold levels for the UK. It should however be noted that oysters placed on the market in the UK generally receive additional commercial processing through depuration (self-purification) in tanks. Further processing is a legal requirement for class B and C oyster production areas and is also frequently applied to class A oysters. Although depuration is generally considered to be relatively ineffective for safeguarding against viral contamination (Doré et al., 1995, Schwab et al., 1998, Muniaín-Mujika et al., 2002) its contribution in reducing norovirus levels has not been systematically assessed, and this area may therefore warrant further investigation. Likewise a study of norovirus levels in oysters placed on the market following commercial processing would help establish the levels of exposure of the UK consumer.

In common with previous studies in Northern Europe (Formiga-Cruz et al., 2002, LeGuyader et al., 2000, Lowther et al., 2008) a very marked seasonality was observed, with highest prevalences and levels during the winter periods. Previously possible contributors to this seasonal pattern of occurrence have been suggested to include; the greater persistence of viral particles under typical Northern European winter environmental conditions of low solar irradiation, low temperature and higher turbidity (Allwood et al., 2005, Cannon et al., 2006); more effective viral clearance in oysters during summer months due to the higher rate of shellfish metabolism in warmer seawaters (Doré et al., 1998, Henroth et al., 2007); and the higher prevalence of noroviruses in the human population, and thus seeded into the marine environment, during the winter (Blanton et al., 2006, Lopman et al., 2003, Mounts et al., 2000). Possibly all are contributory factors, however in this large systematic study the relationship of norovirus to environmental temperature data was found to be stronger than that with numbers of infections reported to national surveillance. Specifically, higher levels of norovirus were found in oysters in the second winter of the study period corresponding to the record low winter temperature but contrasting with a relatively low rate (compared with the first winter period) of lab reports of infections. However this may be due to limits in the surveillance systems currently in place to monitor human norovirus infections; lab reports tend to be related to outbreaks associated with healthcare institutions and may not reflect the overall levels of norovirus in the community.

The findings related to seasonality are consistent with the winter seasonality of norovirus illness incidents following shellfish consumption in epidemiological data both for the UK and for other countries (Doré et al., 2000). During the early months of 2010 an exceptionally large number of incidents of gastroenteritis linked to oysters were reported from restaurants, hotels and similar establishments in the UK. In total for the period July 2009-June 2010, details of 53 clusters of norovirus-type illness associated with consumption of oysters were received by the Cefas Liaison for Outbreaks Associated with Bivalve Molluscs, compared with an average of 10.9 clusters per season (July-June) in 1999-2009 and a previous maximum of 26 clusters in 2005-2006 (personal communication). There were also high numbers of clusters reported in the same period from several other European countries (Westrell et al., 2010); this was linked to unusually severe weather conditions in northern Europe, for example the winter period of 2009-2010 (December to February) was officially the coldest in the United Kingdom since 1978-1979 (Met Office press release 1st March, 2010; http://www.metoffice.gov.uk/news/releases/archive/2010/coldest-uk-winter). On the other hand despite more extreme low temperatures in the following winter (December 2010 was the coldest on record in the UK; Met Office press release 5th January, 2011; http://www.metoffice.gov.uk/news/releases/archive/2011/cold-dec), and the corresponding high levels of norovirus in oysters found in this study, a relatively small number of illness clusters linked to oyster consumption were recorded (11 clusters from July 2010-June 2011; Cefas Liaison for Outbreaks Associated with Bivalve Molluscs, personal communication). It is possible that, following the unusually high number of clusters in the winter of 2009/10, some producers in the UK took steps to lower the risk of illness outbreaks, including voluntary restrictions on harvesting during the winter, and the use of extended depuration times, resulting in a lower number of illness incidents linked to oysters.

In the past many outbreaks of norovirus have been linked to consumption of shellfish that were compliant with bacteriological faecal pollution criteria (Ang, 1998; Chalmers & McMillan, 1995; Gill et al., 1983). However analysis of the relationships between norovirus contamination, and either E. coli levels or classification status in this study offers support for the use of E. coli as an indicator organism for faecal pollution monitoring and risk management purposes. Although on an individual sample basis E. coli was poorly predictive of norovirus risk, on a site-specific basis average E. coli levels were found to correlate with average norovirus scores, particularly when data from the winter only was used. Significant differences were also found in norovirus levels in class A, B and C sites supporting the site specific classification approach. It should however be noted that class B is a very wide category accommodating E. coli levels of between 230 and 46,000 (in 10% of samples) MPN/100g shellfish. Based on the data presented in this study there is clearly scope to further refine the classification categories to improve the correlation with norovirus risk, for example by combining seasonal or temperature-based criteria with E. coli-based classification.

A risk matrix incorporating various weighted factors (population density, E. coli scores, outbreak history etc.) was developed to aid site selection and ensure representative sampling in this study. It is informative that a positive correlation was found between the assigned risk score for each site and the average norovirus levels subsequently determined. Further analysis attempted to refine this risk ranking approach to improve the predictive value for norovirus contamination modelled using the data generated in this study. We have elaborated a risk scoring system based on a combination of the E. coli contamination profile of the site, the site seawater temperature, and the month of sampling. This system performed relatively well for the data generated in this study with, for example, a negative predictive value of 91.3%, although the positive predictive value of 62.2% indicates that it could be further improved. It would be interesting to validate this approach in a selection of areas not monitored during this study. A simple risk scoring matrix based on the principal factors identified here has the potential to focus enhanced risk management measures, such as virus monitoring or enhanced depuration practices, where they are most needed.

In summary this is the first systematic study of norovirus contamination levels in UK oyster production areas and should help inform both the FSA and the producer sector with regard to this risk. It should also assist the FSA with consideration of the impact of possible norovirus threshold risk management levels. 8.0 References

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## Appendix 1: Oyster species comparison results

| GII quantity (copies/g) | O. edulis | C. gigas | |------------------------|-----------|----------| | | extraction 1 | extraction 2 | extraction 1 | extraction 2 | | **High** | | | | | | aliquot 1 | 93454 | 333641 | 141626 | 140236 | | aliquot 2 | 181164 | 240309 | 252699 | 147574 | | aliquot 3 | 142665 | 185666 | 159136 | 291543 | | aliquot 4 | 138860 | 92330 | 104058 | 225560 | | aliquot 5 | 135668 | 207534 | 225154 | 211363 | | **Medium** | | | | | | aliquot 1 | 26099 | 74961 | 26250 | 74309 | | aliquot 2 | 72738 | 168943 | 43008 | 91931 | | aliquot 3 | 32311 | 69099 | 51439 | 117865 | | aliquot 4 | 30796 | 72071 | 44471 | 104847 | | aliquot 5 | 68940 | 181471 | 26764 | 61494 | | **Low** | | | | | | aliquot 1 | 8508 | 6101 | 7227 | 6982 | | aliquot 2 | 16282 | 9318 | 8433 | 3545 | | aliquot 3 | 24900 | 8082 | 10705 | 5396 | | aliquot 4 | 11994 | 7732 | 9213 | 5809 | | aliquot 5 | 10967 | 4913 | 7605 | 3779 |

## Appendix 2: Surveillance sample results

### Site 1

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|---------------| | MAY 09 | 12.5 | \<20 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUN 09 | 15.8 | 50 | not detected | \<40 copies/g | positive | \<100 copies/g | | JUL 09 | 19 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 09 | 20 | 50 | not detected | \<40 copies/g | positive | \<100 copies/g | | SEP 09 | 17 | \<20 | positive | \<100 copies/g | positive | \<100 copies/g | | OCT 09 | | \<20 | positive | \<100 copies/g | not detected | \<40 copies/g | | NOV 09 | 7.8 | 490 | positive | \<100 copies/g | positive | \<100 copies/g | | DEC 09 | 7.5 | 330 | positive | 209 copies/g | positive | 100-500 copies/g | | JAN 10 | 1.6 | 220 | positive | 119 copies/g | positive | 609 copies/g | | FEB 10 | 1.5 | 50 | positive | 739 copies/g | positive | 1914 copies/g | | MAR 10 | 4 | 790 | positive | \<100 copies/g | positive | 1882 copies/g | | APR 10 | 9.2 | \<20 | not detected | \<40 copies/g | positive | 100-500 copies/g | | MAY 10 | 9.8 | 130 | not detected | \<40 copies/g | positive | \<100 copies/g | | JUN 10 | 13.5 | 20 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUL 10 | 19.3 | \<20 | not detected | \<40 copies/g | positive | \<100 copies/g | | AUG 10 | 18 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 10 | 13.5 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | OCT 10 | 10 | 130 | positive | 11741 copies/g| positive | \<100 copies/g | | NOV 10 | 5.5 | 50 | positive | \<100 copies/g | positive | 346 copies/g | | DEC 10 | 1 | 170 | positive | 100-500 copies/g| positive | 658 copies/g | | JAN 11 | 2 | 330 | positive | 260 copies/g | positive | 774 copies/g | | FEB 11 | 5.5 | 700 | positive | \<100 copies/g | positive | 1745 copies/g | | MAR 11 | 3.5 | \<20 | positive | 683 copies/g | positive | 1794 copies/g | | APR 11 | 12 | \<20 | positive | \<100 copies/g | positive | 340 copies/g |

### Site 2

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|--------------| | JUN 09 | 16 | 20 | positive | \<100 copies/g | positive | \<100 copies/g | | JUL 09 | 20.9 | 20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 09 | 21.5 | 50 | positive | \<100 copies/g | positive | 211 copies/g | | SEP 09 | 16.5 | 80 | positive | \<100 copies/g | positive | 170 copies/g | | OCT 09 | 13.5 | 50 | positive | \<100 copies/g | positive | \<100 copies/g | | DEC 09 | 7.5 | 1700 | positive | 334 copies/g | positive | 100-500 copies/g | | JAN 10 | 4.2 | 1700 | positive | \<100 copies/g | positive | 2554 copies/g | | FEB 10 | 2.8 | 490 | positive | 371 copies/g | positive | 5743 copies/g | | MAR 10 | 6.4 | 700 | positive | \<100 copies/g | positive | 4536 copies/g | | APR 10 | 9.9 | 330 | positive | \<100 copies/g | positive | 656 copies/g | | MAY 10 | 10.6 | 70 | positive | \<100 copies/g | positive | 100-500 copies/g | | JUL 10 | 16.5 | 230 | positive | \<100 copies/g | not detected | \<40 copies/g | | AUG 10 | 18.8 | 330 | positive | \<100 copies/g | not detected | \<40 copies/g | | SEP 10 | 15.4 | 260 | positive | \<100 copies/g | positive | \<100 copies/g | | OCT 10 | 9.6 | 170 | positive | 574 copies/g | positive | 928 copies/g | | DEC 10 | \<20 | 1920 | positive | 1116 copies/g | positive | 1990 copies/g | | JAN 11 | 3.2 | 490 | positive | 1116 copies/g | positive | 1990 copies/g | | MAR 11 | 11.5 | 1100 | positive | 13193 copies/g | positive | 5138 copies/g |

### Site 3

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|--------------| | MAY 09 | 14.5 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUN 09 | 18 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 09 | 18.5 | 20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 09 | 16.5 | 20 | positive | \<100 copies/g | not detected | \<40 copies/g | | OCT 09 | 3500 | 50 | not detected | \<40 copies/g | not detected | \<40 copies/g | | NOV 09 | 10.8 | 50 | not detected | \<40 copies/g | not detected | \<40 copies/g | | DEC 09 | 8 | 130 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JAN 10 | 3.2 | 20 | positive | \<100 copies/g | not detected | \<40 copies/g | | FEB 10 | 2.2 | 1700 | positive | \<100 copies/g | not detected | \<40 copies/g | | MAR 10 | 4.8 | 50 | not detected | \<40 copies/g | not detected | \<40 copies/g | | APR 10 | 9.3 | 20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | MAY 10 | 4 | \<20 | not detected | \<100 copies/g | not detected | \<40 copies/g | | JUN 10 | 18 | 50 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 10 | 17 | 80 | positive | \<100 copies/g | not detected | \<40 copies/g | | SEP 10 | 10.8 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | OCT 10 | 6.8 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | NOV 10 | 6 | 80 | positive | \<100 copies/g | not detected | \<40 copies/g | | DEC 10 | 1.5 | 460 | positive | \<100 copies/g | positive | \<100 copies/g | | JAN 11 | 3 | 1700 | positive | 186 copies/g | not detected | \<40 copies/g | | FEB 11 | 6 | \<20 | positive | \<100 copies/g | not detected | \<40 copies/g | | MAR 11 | 8.5 | 330 | positive | \<100 copies/g | positive | \<100 copies/g | | APR 11 | 14 | 20 | positive | \<100 copies/g | not detected | \<40 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 11.3 | 20 | positive | \<100 copies/g | | JUN 09 | 14.3 | \<20 | not detected | \<40 copies/g | | JUL 09 | 18.8 | 110 | not detected | \<40 copies/g | | AUG 09 | 20.2 | 50 | not detected | \<40 copies/g | | SEP 09 | 14.8 | \<20 | positive | \<100 copies/g | | OCT 09 | 13.5 | 50 | positive | \<100 copies/g | | NOV 09 | 8 | \<20 | positive | \<100 copies/g | | DEC 09 | 7.8 | 490 | positive | 203 copies/g | | JAN 10 | 0.8 | 490 | positive | 213 copies/g | | FEB 10 | 2 | 70 | positive | 782 copies/g | | MAR 10 | 1.2 | 3500 | positive | 1168 copies/g | | APR 10 | 8.5 | \<20 | positive | \<100 copies/g | | MAY 10 | 11 | \<20 | positive | \<100 copies/g | | JUN 10 | 12.5 | 20 | positive | \<100 copies/g | | JUL 10 | 18 | 490 | not detected | \<40 copies/g | | AUG 10 | 18 | 80 | not detected | \<40 copies/g | | SEP 10 | 12.2 | 20 | not detected | \<40 copies/g | | OCT 10 | 9.8 | 170 | positive | \<100 copies/g | | NOV 10 | 4.8 | 170 | positive | 100-500 copies/g | | DEC 10 | 2 | 80 | positive | 867 copies/g | | JAN 11 | 4.5 | 790 | positive | 767 copies/g | | FEB 11 | 6.5 | 70 | positive | 133 copies/g | | MAR 11 | 5 | \<20 | positive | 1166 copies/g | | APR 11 | 10 | 80 | positive | 288 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|--------------| | MAY 09 | 80 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUN 09 | 310 | positive | \<100 copies/g | positive | \<100 copies/g | | JUL 09 | 460 | positive | \<100 copies/g | positive | \<100 copies/g | | AUG 09 | 80 | positive | \<100 copies/g | positive | \<100 copies/g | | SEP 09 | 170 | positive | \<100 copies/g | positive | \<100 copies/g | | OCT 09 | 790 | positive | 150 copies/g | positive | 389 copies/g | | NOV 09 | 790 | positive | 180 copies/g | positive | 1012 copies/g | | DEC 09 | 230 | positive | 1042 copies/g | positive | 1981 copies/g | | JAN 10 | 700 | positive | 501 copies/g | positive | 10883 copies/g | | FEB 10 | 170 | positive | 953 copies/g | positive | 3841 copies/g | | MAR 10 | 2400 | positive | 1063 copies/g | positive | 10898 copies/g | | APR 10 | 230 | positive | \<100 copies/g | positive | 136 copies/g | | MAY 10 | 170 | positive | 172 copies/g | positive | 100-500 copies/g | | JUN 10 | 230 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUL 10 | 330 | positive | \<100 copies/g | not detected | \<40 copies/g | | AUG 10 | 80 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 10 | 70 | positive | 278 copies/g | positive | \<100 copies/g | | OCT 10 | 80 | positive | 648 copies/g | positive | \<100 copies/g | | NOV 10 | 790 | positive | 1153 copies/g | positive | 4919 copies/g | | DEC 10 | 790 | positive | 4625 copies/g | positive | 10820 copies/g | | JAN 11 | 330 | positive | 2615 copies/g | positive | 3657 copies/g | | FEB 11 | 330 | positive | 2890 copies/g | positive | 18025 copies/g | | MAR 11 | 790 | positive | 791 copies/g | positive | 1983 copies/g | | APR 11 | 230 | positive | 350 copies/g | positive | 838 copies/g |

### Site 6

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|--------------| | MAY 09 | 20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUN 09 | 1300 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUL 09 | 330 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 09 | 460 | not detected | \<40 copies/g | positive | \<100 copies/g | | SEP 09 | 230 | positive | \<100 copies/g | positive | \<100 copies/g | | NOV 09 | 700 | positive | \<100 copies/g | not detected | \<40 copies/g | | DEC 09 | 230 | positive | \<100 copies/g | not detected | \<40 copies/g | | JAN 10 | 490 | positive | \<100 copies/g | not detected | \<40 copies/g | | FEB 10 | 80 | positive | \<100 copies/g | not detected | \<40 copies/g | | MAR 10 | \<20 | positive | \<100 copies/g | not detected | \<40 copies/g | | APR 10 | 20 | positive | \<100 copies/g | not detected | \<40 copies/g | | MAY 10 | 140 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUN 10 | 230 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 10 | 5400 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 10 | 40 | not detected | \<40 copies/g | not detected | \<40 copies/g | | NOV 10 | 20 | positive | 369 copies/g | not detected | \<40 copies/g | | DEC 10 | 50 | positive | 100-500 copies/g | positive | \<100 copies/g | | JAN 11 | 20 | positive | 100-500 copies/g | positive | \<100 copies/g | | FEB 11 | 80 | positive | \<100 copies/g | positive | 185 copies/g | | MAR 11 | 70 | positive | 172 copies/g | positive | 182 copies/g | | APR 11 | 20 | positive | \<100 copies/g | positive | \<100 copies/g |

### Site 7

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|--------------| | MAY 09 | 10.4 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUN 09 | 10.8 | 80 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 09 | 50 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 09 | 20.4 | 80 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 09 | 17 | 80 | not detected | \<40 copies/g | not detected | \<40 copies/g | | OCT 09 | 16.2 | 170 | positive | \<100 copies/g | positive | \<100 copies/g | | NOV 09 | 9.5 | 1300 | positive | \<100 copies/g | positive | 113 copies/g | | DEC 09 | 8.7 | 790 | positive | 592 copies/g | positive | 405 copies/g | | JAN 10 | 230 | positive | 113 copies/g | positive | 348 copies/g | | FEB 10 | 3 | 490 | positive | \<100 copies/g | positive | 100-500 copies/g | | MAR 10 | 490 | positive | \<100 copies/g | positive | 2548 copies/g | | APR 10 | 10 | 20 | positive | \<100 copies/g | not detected | \<40 copies/g | | MAY 10 | 9.5 | 330 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUN 10 | 170 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUL 10 | 17.6 | 80 | not detected | \<40 copies/g | not detected | \<40 copies/g | | DEC 10 | 3.6 | 170 | positive | \<100 copies/g | positive | \<100 copies/g | | JAN 11 | 5.1 | 230 | positive | 315 copies/g | positive | 3944 copies/g | | FEB 11 | 80 | positive | 220 copies/g | positive | 1465 copies/g | | MAR 11 | 5.2 | 230 | positive | 247 copies/g | positive | 1180 copies/g | | APR 11 | 9 | \<20 | positive | 100-500 copies/g | positive | 279 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 12 | 80 | not detected | \<40 copies/g | | JUN 09 | 15 | 140 | not detected | \<40 copies/g | | JUL 09 | 19 | 2200 | not detected | \<40 copies/g | | AUG 09 | 20 | 3500 | positive | \<100 copies/g | | SEP 09 | 17 | 230 | positive | \<100 copies/g | | OCT 09 | 15 | 40 | not detected | \<40 copies/g | | NOV 09 | 10 | 110 | positive | \<100 copies/g | | DEC 09 | | 130 | positive | \<100 copies/g | | JAN 10 | 3 | 50 | positive | \<100 copies/g | | FEB 10 | 3 | 140 | positive | \<100 copies/g | | MAR 10 | 7 | 50 | positive | \<100 copies/g | | APR 10 | 8 | 310 | positive | \<100 copies/g | | MAY 10 | 11 | \<20 | positive | \<100 copies/g | | JUN 10 | 130 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUL 10 | 19 | 230 | not detected | \<40 copies/g | | AUG 10 | 19 | 790 | not detected | \<40 copies/g | | SEP 10 | 17 | 70 | not detected | \<40 copies/g | | OCT 10 | 15.9 | 790 | not detected | \<40 copies/g | | NOV 10 | 8 | 40 | positive | \<100 copies/g | | DEC 10 | 5 | 220 | not detected | \<40 copies/g | | JAN 11 | 5 | 80 | positive | 115 copies/g | | FEB 11 | 5.8 | 130 | positive | \<100 copies/g | | MAR 11 | 7.5 | \<20 | not detected | \<40 copies/g | | APR 11 | 11 | 20 | positive | \<100 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 12.1 | \<20 | \<100 copies/g| not detected | | JUN 09 | 17 | 20 | \<100 copies/g| not detected | | JUL 09 | 21 | 220 | \<100 copies/g| not detected | | AUG 09 | 18 | 330 | not detected | \<40 copies/g | | SEP 09 | 19 | 50 | not detected | \<40 copies/g | | OCT 09 | 10 | 330 | not detected | \<40 copies/g | | NOV 09 | 12 | 490 | positive | \<100 copies/g | | DEC 09 | 8.5 | 490 | positive | \<100 copies/g | | JAN 10 | 2.9 | 1100 | positive | 1597 copies/g | | FEB 10 | 3.5 | 1300 | positive | \<100 copies/g | | MAR 10 | 6 | 50 | not detected | \<40 copies/g | | APR 10 | 12.5 | \<20 | positive | \<100 copies/g | | MAY 10 | 17 | 50 | positive | \<100 copies/g | | JUN 10 | 17.1 | 80 | positive | \<100 copies/g | | JUL 10 | 20.9 | 70 | positive | \<100 copies/g | | AUG 10 | 19 | 50 | not detected | \<40 copies/g | | SEP 10 | 15 | 50 | positive | \<100 copies/g | | OCT 10 | 12 | 80 | positive | 635 copies/g | | NOV 10 | 6 | 80 | positive | 575 copies/g | | JAN 11 | 6.2 | 3500 | positive | 2043 copies/g | | FEB 11 | 1.8 | 330 | positive | 571 copies/g | | MAR 11 | 4.8 | 130 | positive | 528 copies/g | | APR 11 | 13.2 | 20 | positive | \<100 copies/g |

### Site 10

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|------------------|---------|--------------|--------------| | MAY 09 | 13 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUN 09 | 17.6 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 09 | 19 | \<20 | not detected | \<40 copies/g | positive | \<100 copies/g | | AUG 09 | 20.5 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 09 | 16.5 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | OCT 09 | 14.1 | 20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | NOV 09 | 10.8 | 20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | DEC 09 | 8.7 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JAN 10 | 3.2 | \<20 | positive | \<100 copies/g | not detected | \<40 copies/g | | FEB 10 | 20 | positive | \<100 copies/g | not detected | \<40 copies/g | | MAR 10 | 5.8 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | APR 10 | 9.8 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | MAY 10 | 12.1 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUN 10 | 16.6 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 10 | 20 | 20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 10 | 18.9 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 10 | 17.9 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | OCT 10 | 10.5 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | NOV 10 | 8.5 | 20 | not detected | \<40 copies/g | positive | \<100 copies/g | | DEC 10 | 3.8 | 50 | positive | 171 copies/g | positive | \<100 copies/g | | JAN 11 | 6.2 | 130 | positive | \<100 copies/g | not detected | \<40 copies/g | | FEB 11 | 6.4 | \<20 | positive | \<100 copies/g | not detected | \<40 copies/g | | MAR 11 | 5.6 | \<20 | positive | \<100 copies/g | positive | \<100 copies/g | | APR 11 | 9.8 | \<20 | positive | \<100 copies/g | not detected | \<40 copies/g |

### Site 11

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|------------------|---------|--------------|--------------| | JUN 09 | 16 | 490 | positive | \<100 copies/g | positive | 286 copies/g | | JUL 09 | 20.9 | 3500 | not detected | \<40 copies/g | positive | \<100 copies/g | | SEP 09 | 16.3 | 170 | positive | 741 copies/g | positive | 1163 copies/g | | OCT 09 | 13.1 | 330 | positive | \<100 copies/g | positive | 690 copies/g | | DEC 09 | 7.5 | 1100 | positive | 2190 copies/g | positive | 4949 copies/g | | JAN 10 | 4.2 | 5400 | positive | 887 copies/g | positive | 13272 copies/g | | FEB 10 | 2.8 | 940 | positive | 1124 copies/g | positive | 8380 copies/g | | MAR 10 | 6.4 | 3500 | positive | 149 copies/g | positive | 6923 copies/g | | APR 10 | 9.9 | 460 | not detected | \<100 copies/g | positive | 3190 copies/g | | MAY 10 | 10.6 | 170 | positive | 558 copies/g | positive | 1660 copies/g | | JUL 10 | 16.5 | 1100 | positive | \<100 copies/g | not detected | \<40 copies/g | | AUG 10 | 18.4 | 330 | positive | 378 copies/g | not detected | \<40 copies/g | | SEP 10 | 15.4 | 170 | positive | 1379 copies/g | positive | 459 copies/g | | OCT 10 | 9.6 | 790 | positive | 1649 copies/g | positive | 1287 copies/g | | DEC 10 | \<20 | 4319 | positive | 2496 copies/g | positive | 2496 copies/g | | JAN 11 | 3.5 | 490 | positive | 9385 copies/g | positive | 15369 copies/g | | MAR 11 | 11 | 790 | positive | 16507 copies/g | positive | 5920 copies/g |

### Site 12

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|---------------| | JUN 09 | 15.2 | 1700 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUL 09 | 20.9 | 330 | positive | \<100 copies/g | positive | \<100 copies/g | | AUG 09 | 19 | 170 | positive | \<100 copies/g | positive | \<100 copies/g | | SEP 09 | 17 | 230 | positive | \<100 copies/g | positive | \<100 copies/g | | OCT 09 | 18.5 | 490 | not detected | \<40 copies/g | positive | \<100 copies/g | | NOV 09 | 8.3 | 140 | positive | \<100 copies/g | positive | 100-500 copies/g | | DEC 09 | 6.8 | 490 | positive | 324 copies/g | positive | 2055 copies/g | | FEB 10 | 2.8 | 790 | positive | 255 copies/g | positive | 789 copies/g | | MAR 10 | 6.4 | 230 | positive | 115 copies/g | positive | 5135 copies/g | | APR 10 | 9.9 | 230 | positive | \<100 copies/g | positive | 177 copies/g | | MAY 10 | 10.6 | 330 | positive | 131 copies/g | positive | 798 copies/g | | JUN 10 | 16.5 | 130 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 10 | 18.4 | 230 | positive | \<100 copies/g | not detected | \<40 copies/g | | SEP 10 | 15.2 | 330 | positive | 998 copies/g | positive | 798 copies/g | | OCT 10 | 9.6 | 1300 | positive | 2268 copies/g | positive | 789 copies/g | | JAN 11 | 3.5 | 790 | positive | 1301 copies/g | positive | 3114 copies/g | | MAR 11 | 11.5 | 230 | positive | 1743 copies/g | positive | 1395 copies/g |

### Site 13

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|---------------| | MAY 09 | 2800 | positive | \<100 copies/g | positive | \<100 copies/g | | JUN 09 | 1100 | not detected | \<40 copies/g | positive | \<100 copies/g | | JUL 09 | 2400 | not detected | \<40 copies/g | positive | \<100 copies/g | | AUG 09 | 1700 | positive | \<100 copies/g | positive | \<100 copies/g | | SEP 09 | 3500 | no result | no result | no result | no result | | OCT 09 | 1300 | positive | \<100 copies/g | not detected | \<40 copies/g | | NOV 09 | 8.6 | 2200 | no result | no result | no result | | DEC 09 | 7.4 | 230 | positive | 212 copies/g | positive | 3881 copies/g | | JAN 10 | 4.6 | 700 | positive | 668 copies/g | positive | 1852 copies/g | | FEB 10 | 790 | positive | 123 copies/g | positive | 6127 copies/g | | MAR 10 | 11.5 | 230 | positive | \<100 copies/g | positive | 100-500 copies/g | | APR 10 | 11.7 | 220 | positive | \<100 copies/g | positive | \<100 copies/g | | MAY 10 | 14.2 | 220 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUN 10 | 17.9 | 790 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 10 | 18.4 | 330 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 10 | 9200 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 10 | 18.9 | 5400 | positive | \<100 copies/g | not detected | \<40 copies/g | | OCT 10 | 15.1 | 1100 | positive | 901 copies/g | positive | \<100 copies/g | | NOV 10 | 790 | positive | \<100 copies/g | positive | \<100 copies/g | | DEC 10 | 10 | 490 | positive | \<100 copies/g | positive | 239 copies/g | | JAN 11 | 7.3 | 490 | positive | 479 copies/g | positive | 366 copies/g | | FEB 11 | 13.2 | 1300 | positive | 168 copies/g | positive | 100-500 copies/g | | MAR 11 | 9.3 | 220 | positive | 108 copies/g | positive | 125 copies/g | | APR 11 | 230 | positive | \<100 copies/g | positive | \<100 copies/g |

### Site 14

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|--------------| | MAY 09 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUN 09 | 16 | 20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 09 | 790 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 09 | 1300 | not detected | \<40 copies/g | positive | \<100 copies/g | | SEP 09 | 17 | 490 | positive | \<100 copies/g | positive | 119 copies/g | | OCT 09 | 16 | 130 | positive | \<100 copies/g | not detected | \<40 copies/g | | DEC 09 | 9.5 | 1100 | positive | \<100 copies/g | not detected | \<40 copies/g | | JAN 10 | 4.5 | 5400 | positive | 3190 copies/g | positive | 6249 copies/g | | FEB 10 | 9200 | positive | 926 copies/g | positive | 3957 copies/g | | MAR 10 | 6.5 | 40 | positive | 115 copies/g | positive | 1490 copies/g | | APR 10 | 7.5 | 220 | positive | \<100 copies/g | not detected | \<40 copies/g | | MAY 10 | 10.5 | 50 | positive | \<100 copies/g | positive | \<100 copies/g | | JUN 10 | 15.5 | 80 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 10 | 19.5 | 130 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 10 | 330 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 10 | 17 | 2400 | positive | \<100 copies/g | positive | \<100 copies/g | | OCT 10 | 15 | 16000 | positive | 4794 copies/g | positive | 5042 copies/g |

### Site 15

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|--------------| | MAY 09 | 13.8 | \<20 | positive | \<100 copies/g | positive | \<100 copies/g | | JUN 09 | 17.9 | 110 | no result | no result | no result | no result | | JUL 09 | 18.4 | 80 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 09 | 18.8 | 50 | no result | no result | no result | no result | | SEP 09 | 17.7 | \<20 | positive | \<100 copies/g | not detected | \<40 copies/g | | OCT 09 | 16.3 | 50 | positive | \<100 copies/g | positive | 106 copies/g | | NOV 09 | 9.7 | 20 | positive | \<100 copies/g | positive | 224 copies/g | | DEC 09 | 7.9 | 1300 | positive | \<100 copies/g | positive | 326 copies/g | | JAN 10 | 5.3 | 330 | positive | 179 copies/g | positive | 1123 copies/g | | FEB 10 | 230 | positive | 230 copies/g | positive | 637 copies/g | | MAR 10 | 170 | positive | \<100 copies/g | positive | 964 copies/g | | APR 10 | 10.5 | 230 | positive | 155 copies/g | positive | 854 copies/g | | MAY 10 | 11.9 | 130 | positive | \<100 copies/g | positive | \<100 copies/g | | JUN 10 | 15 | 230 | positive | \<100 copies/g | positive | \<100 copies/g | | JUL 10 | 19.8 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 10 | 18.3 | \<20 | not detected | \<100 copies/g | not detected | \<40 copies/g | | SEP 10 | 17.4 | 790 | not detected | \<40 copies/g | not detected | \<40 copies/g | | OCT 10 | 16.5 | 700 | positive | 381 copies/g | positive | \<100 copies/g | | NOV 10 | 20 | positive | \<100 copies/g | positive | \<100 copies/g | | DEC 10 | 5 | 220 | positive | \<100 copies/g | positive | 513 copies/g | | JAN 11 | 5.4 | 460 | positive | \<100 copies/g | positive | \<100 copies/g | | FEB 11 | 4.5 | 130 | positive | \<100 copies/g | positive | 312 copies/g | | MAR 11 | 130 | positive | \<100 copies/g | positive | \<100 copies/g | | APR 11 | 12 | 490 | positive | 192 copies/g | positive | 100-500 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|--------------| | MAY 09 | 13.1 | 490 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUN 09 | 16.4 | 460 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 09 | 18.5 | 490 | positive | \<100 copies/g | not detected | \<40 copies/g | | AUG 09 | 19.6 | 460 | positive | \<100 copies/g | positive | \<100 copies/g | | SEP 09 | 16.9 | 220 | positive | \<100 copies/g | positive | \<100 copies/g | | OCT 09 | 14.9 | 790 | positive | \<100 copies/g | positive | 100-500 copies/g | | NOV 09 | 11.8 | 5400 | positive | \<100 copies/g | not detected | \<40 copies/g | | DEC 09 | 10 | 230 | positive | \<100 copies/g | positive | 143 copies/g | | JAN 10 | 4.7 | 260 | positive | \<100 copies/g | positive | 100-500 copies/g | | FEB 10 | 5.6 | 80 | positive | \<100 copies/g | positive | \<100 copies/g | | MAR 10 | 5.5 | 170 | positive | \<100 copies/g | positive | 569 copies/g | | APR 10 | 230 | positive | \<100 copies/g | not detected | \<40 copies/g | | MAY 10 | 14 | 50 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUN 10 | 17.3 | 230 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUL 10 | 19.4 | 2400 | positive | \<100 copies/g | not detected | \<40 copies/g | | AUG 10 | 18.6 | 330 | positive | 100-500 copies/g | not detected | \<40 copies/g | | OCT 10 | 15.3 | 330 | positive | 664 copies/g | positive | \<100 copies/g | | NOV 10 | 10.8 | 3500 | positive | 401 copies/g | positive | 1640 copies/g | | DEC 10 | 5.8 | 490 | positive | 884 copies/g | positive | 1225 copies/g | | JAN 11 | 6.8 | 1700 | positive | 488 copies/g | positive | 1401 copies/g | | FEB 11 | 7.1 | 330 | positive | 531 copies/g | positive | 558 copies/g | | MAR 11 | 8 | 1300 | positive | 383 copies/g | positive | 1118 copies/g | | APR 11 | 6.5 | 20 | positive | 865 copies/g | positive | 100-500 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|-------------|--------------|---------------| | MAY 09 | 13.2 | not detected| \<40 copies/g | positive | | JUN 09 | 16.2 | positive | \<100 copies/g| positive | | JUL 09 | 18.1 | not detected| \<40 copies/g | not detected | | AUG 09 | 19.7 | positive | \<100 copies/g| not detected | | SEP 09 | 17 | not detected| \<40 copies/g | positive | | OCT 09 | 15.3 | positive | \<100 copies/g| positive | | NOV 09 | 11.8 | positive | \<100 copies/g| not detected | | DEC 09 | 10.7 | positive | 240 copies/g | not detected | | JAN 10 | 5.3 | positive | 102 copies/g | positive | | FEB 10 | 4.6 | positive | 489 copies/g | positive | | MAR 10 | 5.7 | positive | \<100 copies/g| positive | | APR 10 | 230 | positive | \<100 copies/g| positive | | MAY 10 | 14 | positive | \<100 copies/g| not detected | | JUN 10 | 16.6 | not detected| \<40 copies/g | not detected | | JUL 10 | 19.4 | not detected| \<40 copies/g | not detected | | AUG 10 | 18.3 | positive | \<100 copies/g| not detected | | OCT 10 | 40 | positive | 340 copies/g | positive | | NOV 10 | 11.2 | positive | \<100 copies/g| positive | | DEC 10 | 6 | positive | 100-500 copies/g | positive | | JAN 11 | 6.8 | positive | 146 copies/g | positive | | FEB 11 | 7 | positive | 161 copies/g | positive | | MAR 11 | 220 | positive | 208 copies/g | positive | | APR 11 | 80 | positive | 251 copies/g | positive | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |--------|-------------------|---------|--------------|---------------| | MAY 09 | 13.6 | 170 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUN 09 | 19.1 | \<20 | positive | \<100 copies/g | positive | \<100 copies/g | | JUL 09 | 16.3 | 700 | positive | \<100 copies/g | positive | \<100 copies/g | | AUG 09 | 17.5 | 1300 | positive | \<100 copies/g | positive | \<100 copies/g | | SEP 09 | 16.7 | 110 | positive | \<100 copies/g | not detected | \<40 copies/g | | OCT 09 | 130 | positive| \<100 copies/g| not detected | \<40 copies/g | | NOV 09 | \<20 | positive| \<100 copies/g| positive | \<100 copies/g | | DEC 09 | 10.1 | 50 | positive | 195 copies/g | positive | \<100 copies/g | | JAN 10 | \<20 | positive| 487 copies/g | positive | 512 copies/g | | FEB 10 | \<20 | positive| \<100 copies/g| positive | 1223 copies/g | | MAR 10 | 6.1 | 20 | not detected | \<40 copies/g | positive | 164 copies/g | | APR 10 | 11.5 | 20 | positive | \<100 copies/g | positive | \<100 copies/g | | MAY 10 | 13.2 | 170 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUN 10 | 16.8 | 790 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 10 | 15.1 | 230 | positive | \<100 copies/g | not detected | \<40 copies/g | | AUG 10 | 17.3 | 20 | positive | \<100 copies/g | not detected | \<40 copies/g | | SEP 10 | 130 | positive| \<100 copies/g| positive | \<100 copies/g | | OCT 10 | 3500 | positive| 322 copies/g | positive | 340 copies/g | | NOV 10 | 70 | positive| \<100 copies/g| positive | \<100 copies/g | | DEC 10 | 20 | positive| 331 copies/g | positive | 1468 copies/g | | JAN 11 | 8.1 | \<20 | positive | \<100 copies/g | positive | 1312 copies/g | | FEB 11 | 9 | 490 | positive | 786 copies/g | positive | 1158 copies/g | | MAR 11 | 230 | positive| 202 copies/g | positive | 835 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|--------------| | MAY 09 | 50 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUN 09 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 09 | 50 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 09 | 790 | positive | \<100 copies/g | not detected | \<40 copies/g | | SEP 09 | 80 | not detected | \<40 copies/g | positive | \<100 copies/g | | OCT 09 | 80 | positive | \<100 copies/g | not detected | \<40 copies/g | | DEC 09 | 2400 | positive | 1137 copies/g | positive | 905 copies/g | | JAN 10 | 130 | positive | \<100 copies/g | positive | \<100 copies/g | | FEB 10 | 170 | not detected | \<40 copies/g | positive | \<100 copies/g | | MAR 10 | \<20 | not detected | \<40 copies/g | positive | \<100 copies/g | | APR 10 | \<20 | positive | \<100 copies/g | not detected | \<40 copies/g | | MAY 10 | \<20 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUN 10 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 10 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 10 | \<20 | positive | \<100 copies/g | not detected | \<40 copies/g | | SEP 10 | 1700 | not detected | \<40 copies/g | not detected | \<40 copies/g | | NOV 10 | 50 | not detected | \<40 copies/g | not detected | \<40 copies/g | | DEC 10 | 330 | positive | \<100 copies/g | positive | 100-500 copies/g | | JAN 11 | 330 | positive | \<100 copies/g | positive | 465 copies/g | | FEB 11 | 130 | not detected | \<40 copies/g | positive | 185 copies/g | | MAR 11 | 330 | positive | \<100 copies/g | positive | 4089 copies/g | | APR 11 | 700 | not detected | \<40 copies/g | not detected | \<40 copies/g |

### Site 20

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|--------------| | JUN 09 | 790 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 09 | 2400 | positive | \<100 copies/g | not detected | \<40 copies/g | | AUG 09 | 2400 | positive | \<100 copies/g | not detected | \<40 copies/g | | SEP 09 | 3500 | positive | \<100 copies/g | positive | \<100 copies/g | | OCT 09 | 1300 | positive | \<100 copies/g | positive | \<100 copies/g | | NOV 09 | 8.6 | 490 | no result | no result | | | DEC 09 | 7.4 | 330 | positive | \<100 copies/g | positive | 226 copies/g | | JAN 10 | 4.6 | 330 | positive | 191 copies/g | positive | 1043 copies/g | | FEB 10 | 490 | positive | \<100 copies/g | positive | 933 copies/g | | MAR 10 | 11.5 | 330 | positive | \<100 copies/g | positive | 838 copies/g | | APR 10 | 11.7 | 330 | positive | \<100 copies/g | positive | 100-500 copies/g | | MAY 10 | 14.2 | 70 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUN 10 | 17.9 | 790 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 10 | 18.4 | 1700 | positive | \<100 copies/g | positive | \<100 copies/g | | AUG 10 | 2400 | positive | \<100 copies/g | not detected | \<40 copies/g | | SEP 10 | 18.9 | 3500 | positive | 192 copies/g | not detected | \<40 copies/g | | OCT 10 | 15.1 | 790 | positive | \<100 copies/g | positive | \<100 copies/g | | NOV 10 | 790 | positive | \<100 copies/g | positive | \<100 copies/g | | DEC 10 | 10 | 790 | positive | 118 copies/g | positive | 320 copies/g | | JAN 11 | 7.3 | 460 | positive | 445 copies/g | positive | 517 copies/g | | FEB 11 | 13.2 | 330 | positive | \<100 copies/g | positive | 100-500 copies/g | | MAR 11 | 9.3 | 490 | positive | 115 copies/g | positive | \<100 copies/g | | APR 11 | 790 | positive | \<100 copies/g | not detected | \<40 copies/g |

### Site 21

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|--------------| | AUG 09 | 14.3 | 460 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 09 | 14.6 | 790 | positive | \<100 copies/g | positive | \<100 copies/g | | OCT 09 | 15.4 | 330 | no result | no result | no result | | | DEC 09 | 8.5 | 110 | positive | \<100 copies/g | not detected | \<40 copies/g | | JAN 10 | 7.8 | 1100 | positive | 278 copies/g | positive | 100-500 copies/g | | FEB 10 | 7.3 | \<20 | positive | \<100 copies/g | positive | \<100 copies/g | | MAR 10 | 6.7 | \<20 | positive | \<100 copies/g | not detected | \<40 copies/g | | MAY 10 | 11.3 | 20 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUN 10 | 13.5 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 10 | 15.6 | 330 | positive | \<100 copies/g | not detected | \<40 copies/g | | AUG 10 | 15.9 | 230 | positive | \<100 copies/g | not detected | \<40 copies/g | | NOV 10 | 13.1 | 9200 | positive | \<100 copies/g | positive | 743 copies/g | | DEC 10 | 8.7 | 1700 | positive | \<100 copies/g | positive | 534 copies/g | | JAN 11 | 8.5 | 330 | positive | 720 copies/g | positive | 655 copies/g | | FEB 11 | 80 | positive | 296 copies/g | positive | 4075 copies/g | | MAR 11 | 9 | 50 | positive | \<100 copies/g | positive | 124 copies/g |

### Site 22

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|--------------| | MAY 09 | 20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUN 09 | 330 | no result | | no result | | | JUL 09 | 330 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 09 | 310 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 09 | 11.8 | 80 | not detected | \<40 copies/g | not detected | \<40 copies/g | | OCT 09 | 14 | 130 | positive | \<100 copies/g | not detected | \<40 copies/g | | NOV 09 | 11 | 170 | positive | \<100 copies/g | positive | \<100 copies/g | | DEC 09 | 8 | 70 | positive | 114 copies/g | positive | 605 copies/g | | JAN 10 | 4 | 130 | positive | \<100 copies/g | positive | 100-500 copies/g | | FEB 10 | 7 | 20 | positive | \<100 copies/g | positive | 100-500 copies/g | | MAR 10 | 8 | 130 | positive | \<100 copies/g | not detected | \<40 copies/g | | APR 10 | 8 | 230 | positive | \<100 copies/g | not detected | \<40 copies/g | | MAY 10 | 10 | 70 | not detected | \<40 copies/g | positive | \<100 copies/g | | JUN 10 | 14 | 490 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 10 | 18 | 80 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 10 | 18 | 130 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 10 | 17 | 50 | not detected | \<40 copies/g | not detected | \<40 copies/g | | OCT 10 | 15 | 9200 | not detected | \<40 copies/g | not detected | \<40 copies/g | | NOV 10 | 13 | 490 | positive | \<100 copies/g | not detected | \<40 copies/g | | DEC 10 | 7 | 210 | positive | \<100 copies/g | positive | 1967 copies/g | | JAN 11 | 7 | 130 | not detected | \<40 copies/g | positive | 769 copies/g | | FEB 11 | 7 | 50 | not detected | \<40 copies/g | positive | 392 copies/g | | MAR 11 | 7 | 70 | not detected | \<40 copies/g | positive | 669 copies/g | | APR 11 | 40 | 40 | not detected | \<40 copies/g | not detected | \<40 copies/g |

### Site 23

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|--------------| | MAY 09 | 310 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUN 09 | 17 | 2800 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 09 | 18.5 | 790 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 09 | 790 | positive | \<100 copies/g | positive | \<100 copies/g | | SEP 09 | 17 | 330 | positive | \<100 copies/g | positive | \<100 copies/g | | OCT 09 | 16 | 330 | positive | \<100 copies/g | not detected | \<40 copies/g | | DEC 09 | 9.5 | 2200 | positive | 1311 copies/g | positive | 4736 copies/g | | JAN 10 | 4.5 | 2400 | positive | \<100 copies/g | positive | 294 copies/g | | FEB 10 | 5 | 330 | positive | \<100 copies/g | positive | 630 copies/g | | MAR 10 | 6.5 | 490 | positive | \<100 copies/g | positive | 1088 copies/g | | APR 10 | 7.5 | 460 | positive | \<100 copies/g | not detected | \<40 copies/g | | MAY 10 | 10 | 16000 | positive | \<100 copies/g | positive | \<100 copies/g | | JUN 10 | 15.5 | 230 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 10 | 19.5 | 80 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 10 | 19 | 230 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 10 | 17 | 330 | not detected | \<40 copies/g | not detected | \<40 copies/g | | OCT 10 | 15 | 3500 | positive | 100-500 copies/g | positive | 100-500 copies/g |

### Site 24

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|---------------| | JUL 09 | 15.6 | 9200 | positive | \<100 copies/g | positive | 140 copies/g | | AUG 09 | 15.8 | 790 | positive | \<100 copies/g | positive | 137 copies/g | | SEP 09 | 14.8 | 1700 | positive | 237 copies/g | positive | 206 copies/g | | OCT 09 | 14.5 | 230 | positive | \<100 copies/g | positive | \<100 copies/g | | NOV 09 | 12.2 | 940 | positive | \<100 copies/g | not detected | \<40 copies/g | | DEC 09 | 10.4 | 1700 | positive | 165 copies/g | positive | \<100 copies/g | | JAN 10 | 7.3 | 170 | positive | \<100 copies/g | positive | \<100 copies/g | | FEB 10 | 7 | 490 | positive | 151 copies/g | positive | \<100 copies/g | | MAR 10 | 7.9 | 230 | positive | \<100 copies/g | positive | 798 copies/g | | APR 10 | 8.8 | 790 | positive | \<100 copies/g | not detected | \<40 copies/g | | MAY 10 | 10.3 | 940 | not detected | \<40 copies/g | positive | \<100 copies/g | | JUN 10 | 15.4 | 3500 | positive | \<100 copies/g | positive | 297 copies/g | | JUL 10 | 10 | 490 | positive | 100-500 copies/g | not detected | \<40 copies/g | | AUG 10 | 16.6 | 230 | positive | 548 copies/g | not detected | \<40 copies/g | | SEP 10 | 17 | 270 | positive | 510 copies/g | positive | \<100 copies/g | | OCT 10 | 15.2 | 2400 | positive | 757 copies/g | positive | 571 copies/g | | NOV 10 | 13.5 | 5400 | positive | 504 copies/g | positive | 1108 copies/g | | DEC 10 | 7.5 | 170 | positive | 423 copies/g | positive | 1561 copies/g | | FEB 11 | 8.9 | 330 | positive | 109 copies/g | positive | 657 copies/g | | MAR 11 | 8.3 | 1300 | positive | \<100 copies/g | positive | 1112 copies/g |

### Site 25

| Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|-------------------|---------|--------------|---------------| | MAY 09 | 13 | 490 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUN 09 | 18 | 20 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUL 09 | 19 | 210 | positive | \<100 copies/g | positive | \<100 copies/g | | AUG 09 | 20 | 3500 | positive | \<100 copies/g | not detected | \<40 copies/g | | SEP 09 | 18 | 170 | positive | \<100 copies/g | positive | 109 copies/g | | NOV 09 | 13 | 2400 | positive | \<100 copies/g | positive | 637 copies/g | | DEC 09 | 9 | 1100 | positive | 209 copies/g | positive | \<100 copies/g | | JAN 10 | 4 | 3500 | positive | 218 copies/g | positive | 3638 copies/g | | FEB 10 | 4 | 1300 | positive | 361 copies/g | positive | 3452 copies/g | | MAR 10 | 4 | 2400 | positive | 639 copies/g | positive | 3505 copies/g | | APR 10 | 8 | 330 | positive | 286 copies/g | positive | 157 copies/g | | AUG 10 | 18 | 80 | positive | 139 copies/g | positive | 357 copies/g | | SEP 10 | 15 | 330 | not detected | \<40 copies/g | not detected | \<40 copies/g | | NOV 10 | 11 | 5400 | positive | 100-500 copies/g | positive | 739 copies/g | | DEC 10 | 3 | 130 | positive | 1178 copies/g | positive | 466 copies/g | | JAN 11 | 3 | 130 | positive | 186 copies/g | positive | 235 copies/g | | FEB 11 | 8 | 170 | positive | 286 copies/g | positive | 2606 copies/g | | MAR 11 | 12 | 110 | positive | 151 copies/g | positive | 575 copies/g | | APR 11 | 13 | 140 | positive | 519 copies/g | positive | 118 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 13 | 50 | not detected | \<40 copies/g | | | | | not detected | \<40 copies/g | | JUN 09 | 8 | 230 | positive | \<100 copies/g | | | | | not detected | \<40 copies/g | | JUL 09 | 16 | 330 | positive | \<100 copies/g | | | | | positive | \<100 copies/g | | AUG 09 | 12 | 1300 | not detected | \<40 copies/g | | | | | positive | \<100 copies/g | | SEP 09 | 10 | 330 | not detected | \<40 copies/g | | | | | positive | \<100 copies/g | | OCT 09 | 10 | 2400 | not detected | \<40 copies/g | | | | | positive | \<100 copies/g | | DEC 09 | 9 | 330 | positive | \<100 copies/g | | | | | not detected | \<40 copies/g | | JAN 10 | 2 | 50 | positive | \<100 copies/g | | | | | positive | \<100 copies/g | | FEB 10 | 4 | 1300 | positive | \<100 copies/g | | | | | positive | 100-500 copies/g | | MAR 10 | 6 | 90 | positive | \<100 copies/g | | | | | positive | 286 copies/g | | APR 10 | 7 | 490 | not detected | \<40 copies/g | | | | | positive | \<100 copies/g | | MAY 10 | 10 | 130 | not detected | \<40 copies/g | | | | | not detected | \<40 copies/g | | JUN 10 | 11 | 70 | not detected | \<40 copies/g | | | | | not detected | \<40 copies/g | | JUL 10 | 11 | 70 | positive | \<100 copies/g | | | | | not detected | \<40 copies/g | | AUG 10 | 12 | 230 | positive | \<100 copies/g | | | | | not detected | \<40 copies/g | | SEP 10 | 10 | 790 | positive | \<100 copies/g | | | | | not detected | \<40 copies/g | | OCT 10 | 9 | 130 | positive | \<100 copies/g | | | | | positive | \<100 copies/g | | NOV 10 | | 490 | positive | 247 copies/g | | | | | positive | 669 copies/g | | DEC 10 | -1 | 50 | positive | 506 copies/g | | | | | positive | \<100 copies/g | | JAN 11 | 6 | 110 | positive | \<100 copies/g | | | | | positive | \<100 copies/g | | FEB 11 | 5 | 130 | positive | 344 copies/g | | | | | positive | 872 copies/g | | MAR 11 | 4 | \<20 | positive | 402 copies/g | | | | | positive | 332 copies/g | | APR 11 | 10 | \<20 | positive | \<100 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 11 | \<20 | not detected | \<40 copies/g | | JUN 09 | 13 | 20 | not detected | \<40 copies/g | | JUL 09 | 12 | 330 | not detected | \<40 copies/g | | AUG 09 | 1100 | positive| \<100 copies/g| | SEP 09 | 10 | 230 | positive | \<100 copies/g | | OCT 09 | 11 | 330 | positive | \<100 copies/g | | NOV 09 | 14 | 330 | positive | \<100 copies/g | | DEC 09 | 12 | 80 | positive | \<100 copies/g | | JAN 10 | 9 | 80 | positive | 120 copies/g | | FEB 10 | 8 | \<20 | positive | 102 copies/g | | MAR 10 | 7 | 110 | positive | \<100 copies/g | | APR 10 | 8 | \<20 | not detected | \<40 copies/g | | MAY 10 | 14 | \<20 | positive | \<100 copies/g | | JUN 10 | 14 | 80 | positive | \<100 copies/g | | JUL 10 | 17 | 50 | not detected | \<40 copies/g | | AUG 10 | 17 | 170 | not detected | \<40 copies/g | | SEP 10 | 17 | 330 | not detected | \<40 copies/g | | OCT 10 | 15 | 80 | positive | \<100 copies/g | | NOV 10 | 13 | 1300 | not detected | \<40 copies/g | | DEC 10 | 11 | 50 | positive | \<100 copies/g | | JAN 11 | 10 | 130 | positive | 317 copies/g | | FEB 11 | 9 | 80 | positive | 197 copies/g | | MAR 11 | 9 | \<20 | positive | \<100 copies/g | | APR 11 | 9.6 | 50 | positive | \<100 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 11 | 50 | not detected | \<40 copies/g | | | | | | not detected | | JUN 09 | 13 | 230 | not detected | \<40 copies/g | | | | | | positive | | JUL 09 | 12 | 5400 | positive | \<100 copies/g | | | | | | positive | | AUG 09 | 17 | 330 | positive | \<100 copies/g | | | | | | not detected | | SEP 09 | 16 | 1100 | positive | \<100 copies/g | | | | | | positive | | OCT 09 | 11 | \<20 | positive | \<100 copies/g | | | | | | not detected | | NOV 09 | 14 | 5400 | positive | \<100 copies/g | | | | | | positive | | DEC 09 | 12 | 130 | positive | \<100 copies/g | | | | | | positive | | JAN 10 | 9 | 460 | positive | \<100 copies/g | | | | | | positive | | FEB 10 | 8 | 50 | positive | \<100 copies/g | | | | | | positive | | MAR 10 | 13 | 130 | positive | \<100 copies/g | | | | | | not detected | | APR 10 | 8 | 130 | not detected | \<40 copies/g | | | | | | not detected | | MAY 10 | 14 | 220 | positive | \<100 copies/g | | | | | | not detected | | JUL 10 | 17 | 170 | not detected | \<40 copies/g | | | | | | not detected | | AUG 10 | 17 | 490 | not detected | \<40 copies/g | | | | | | not detected | | SEP 10 | 16 | 230 | not detected | \<40 copies/g | | | | | | not detected | | OCT 10 | 15 | 2200 | not detected | \<40 copies/g | | | | | | not detected | | NOV 10 | 13 | 1700 | positive | \<100 copies/g | | | | | | not detected | | DEC 10 | 11 | 80 | positive | \<100 copies/g | | | | | | positive | | JAN 11 | 10 | 490 | positive | 130 copies/g | | | | | | positive | | FEB 11 | 9 | 80 | positive | \<100 copies/g | | | | | | positive | | MAR 11 | 9 | 20 | positive | \<100 copies/g | | | | | | positive | | APR 11 | 9.6 | \<20 | positive | \<100 copies/g | | | | | | not detected | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 20 | positive| \<100 copies/g| not detected | | JUN 09 | 15.1 | not detected| \<40 copies/g| not detected | | JUL 09 | 19.9 | positive| \<100 copies/g| not detected | | AUG 09 | 230 | positive| \<100 copies/g| not detected | | SEP 09 | 16.1 | positive| \<100 copies/g| positive | | OCT 09 | 15.3 | positive| \<100 copies/g| positive | | NOV 09 | 10.1 | positive| \<100 copies/g| positive | | DEC 09 | 8.5 | positive| 109 copies/g | positive | | JAN 10 | 4 | positive| \<100 copies/g| positive | | FEB 10 | 5.6 | positive| \<100 copies/g| positive | | MAR 10 | 8.9 | \<20 | not detected| \<40 copies/g | | APR 10 | 11.5 | positive| \<100 copies/g| positive | | MAY 10 | 16.2 | not detected| \<40 copies/g| not detected | | JUN 10 | 18 | positive| \<100 copies/g| not detected | | JUL 10 | 21 | positive| \<100 copies/g| positive | | AUG 10 | 17.5 | \<20 | positive | \<100 copies/g | | SEP 10 | 15.2 | not detected| \<40 copies/g| not detected | | OCT 10 | 50 | not detected| \<40 copies/g| positive | | NOV 10 | 6 | positive| 265 copies/g | positive | | DEC 10 | 6.3 | positive| 505 copies/g | positive | | JAN 11 | 6.1 | positive| 238 copies/g | positive | | FEB 11 | 7.2 | \<20 | positive | 287 copies/g | | MAR 11 | 10 | \<20 | positive | \<40 copies/g | | APR 11 | 10 | positive| \<100 copies/g| not detected | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 11 | \<20 | not detected | \<40 copies/g | | JUN 09 | 13 | 460 | not detected | \<40 copies/g | | JUL 09 | 12 | 3500 | positive | \<100 copies/g | | AUG 09 | 17 | 790 | positive | \<100 copies/g | | SEP 09 | 16 | 330 | positive | \<100 copies/g | | OCT 09 | 11 | 80 | positive | \<100 copies/g | | NOV 09 | 14 | 790 | positive | 132 copies/g | | DEC 09 | 12 | 230 | positive | 184 copies/g | | JAN 10 | 9 | 630 | positive | \<100 copies/g | | FEB 10 | 8 | 130 | positive | \<100 copies/g | | MAR 10 | 20 | 20 | positive | \<100 copies/g | | APR 10 | 8 | 340 | not detected | \<40 copies/g | | MAY 10 | 14 | 330 | not detected | \<40 copies/g | | JUN 10 | 14 | 130 | not detected | \<40 copies/g | | JUL 10 | 17 | 490 | not detected | \<40 copies/g | | AUG 10 | 17 | 1100 | positive | \<100 copies/g | | SEP 10 | 16 | 2400 | not detected | \<40 copies/g | | OCT 10 | 15 | 460 | not detected | \<40 copies/g | | NOV 10 | 13 | 230 | not detected | \<40 copies/g | | DEC 10 | 11 | 230 | positive | \<100 copies/g | | JAN 11 | 10 | 490 | positive | 138 copies/g | | FEB 11 | 9 | 20 | positive | \<100 copies/g | | MAR 11 | 9 | \<20 | positive | \<100 copies/g | | APR 11 | 9.6 | 20 | positive | \<100 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 12 | \<20 | not detected | \<40 copies/g | | JUN 09 | 12.8 | \<20 | not detected | \<40 copies/g | | JUL 09 | 14 | 130 | not detected | \<40 copies/g | | AUG 09 | 14 | 3500 | positive | \<100 copies/g | | SEP 09 | 14 | 490 | no result | no result | | OCT 09 | 13 | 230 | positive | \<100 copies/g | | NOV 09 | 12 | 330 | positive | 417 copies/g | | DEC 09 | 11 | 20 | positive | 1240 copies/g | | JAN 10 | 11 | \<20 | positive | 1140 copies/g | | FEB 10 | 6 | \<20 | positive | 626 copies/g | | MAR 10 | 8.1 | 20 | positive | 100-500 copies/g | | APR 10 | 10.1 | \<20 | not detected | \<100 copies/g | | MAY 10 | 11 | \<20 | not detected | \<40 copies/g | | JUN 10 | 12 | 50 | no result | no result | | JUL 10 | 13 | \<20 | not detected | \<100 copies/g | | AUG 10 | 15 | 40 | not detected | \<40 copies/g | | SEP 10 | 13 | 20 | positive | 100-500 copies/g | | OCT 10 | 10 | \<20 | positive | \<100 copies/g | | NOV 10 | 8 | 20 | positive | 126 copies/g | | DEC 10 | 1 | 80 | positive | 474 copies/g | | JAN 11 | 8 | \<20 | positive | \<100 copies/g | | FEB 11 | 20 | 204 | positive | 586 copies/g | | MAR 11 | \<20 | 157 | positive | 100-500 copies/g | | APR 11 | \<20 | \<100 | not detected | \<40 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |-------|------------------|---------|--------------|--------------| | MAY 09 | 12 | 2400 | positive | \<100 copies/g | positive | \<100 copies/g | | JUN 09 | 12 | 50 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUL 09 | 12 | 490 | positive | \<100 copies/g | positive | \<100 copies/g | | AUG 09 | 12 | 460 | positive | \<100 copies/g | positive | \<100 copies/g | | SEP 09 | 12 | 16000 | positive | \<100 copies/g | positive | \<100 copies/g | | OCT 09 | 12 | 490 | positive | 393 copies/g | positive | 751 copies/g | | DEC 09 | 12 | 80 | positive | 218 copies/g | positive | 100-500 copies/g | | MAR 10 | 7 | 170 | positive | \<100 copies/g | positive | \<100 copies/g | | APR 10 | 7 | 790 | positive | \<100 copies/g | positive | \<100 copies/g | | MAY 10 | 11 | \<20 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUN 10 | 13 | 170 | positive | \<100 copies/g | not detected | \<40 copies/g | | JUL 10 | 14 | 700 | positive | 147 copies/g | not detected | \<40 copies/g | | AUG 10 | 14 | 1100 | positive | 124 copies/g | not detected | \<40 copies/g | | SEP 10 | 12 | 80 | positive | 100-500 copies/g | not detected | \<40 copies/g | | OCT 10 | | 790 | positive | 199 copies/g | positive | \<100 copies/g | | NOV 10 | 12 | 790 | positive | 278 copies/g | positive | 270 copies/g | | DEC 10 | 11 | 790 | positive | 1625 copies/g | positive | 743 copies/g | | JAN 11 | 10 | void | positive | 256 copies/g | positive | \<100 copies/g | | FEB 11 | 10 | 70 | positive | \<100 copies/g | positive | 203 copies/g | | APR 11 | 10 | 50 | positive | 256 copies/g | positive | 306 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 11.6 | 130 | positive | \<100 copies/g | | JUN 09 | 17.2 | 2400 | positive | \<100 copies/g | | JUL 09 | 17.5 | 700 | not detected | \<40 copies/g | | AUG 09 | 17 | 130 | no result | no result | | SEP 09 | 15.6 | 330 | positive | 224 copies/g | | OCT 09 | 14.8 | 490 | positive | \<100 copies/g | | NOV 09 | 10.7 | 460 | positive | \<100 copies/g | | DEC 09 | 8 | 2400 | positive | \<100 copies/g | | JAN 10 | 4 | 80 | positive | 126 copies/g | | FEB 10 | 5 | 490 | positive | 2952 copies/g | | MAR 10 | 4.5 | 110 | positive | 214 copies/g | | APR 10 | 11 | 490 | positive | \<100 copies/g | | MAY 10 | 16.9 | 790 | positive | \<100 copies/g | | JUN 10 | 13.2 | 1300 | positive | \<100 copies/g | | JUL 10 | 17 | 1700 | not detected | \<40 copies/g | | SEP 10 | 16.1 | 460 | positive | \<100 copies/g | | OCT 10 | 12.9 | 330 | positive | 359 copies/g | | NOV 10 | 12 | 2400 | positive | 354 copies/g | | DEC 10 | 5 | 790 | positive | 2356 copies/g | | FEB 11 | 7.7 | 310 | positive | 746 copies/g | | MAR 11 | 8.4 | 230 | positive | 1133 copies/g | | APR 11 | 10.5 | 230 | positive | 146 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 13 | \<20 | not detected | \<40 copies/g | | JUN 09 | 16 | 80 | not detected | \<40 copies/g | | JUL 09 | 15 | 9200 | not detected | \<40 copies/g | | AUG 09 | 15 | 490 | not detected | \<40 copies/g | | SEP 09 | 14 | 230 | positive | \<100 copies/g | | OCT 09 | 9 | 230 | not detected | \<40 copies/g | | NOV 09 | 11 | 1300 | not detected | \<40 copies/g | | DEC 09 | 8 | 230 | not detected | \<40 copies/g | | JAN 10 | 2 | 20 | not detected | \<40 copies/g | | FEB 10 | 2 | \<20 | positive | \<100 copies/g | | MAR 10 | 3 | \<20 | not detected | \<40 copies/g | | APR 10 | 9 | 20 | positive | \<100 copies/g | | MAY 10 | 13 | \<20 | not detected | \<40 copies/g | | JUN 10 | 17 | 50 | positive | \<100 copies/g | | JUL 10 | 16 | 1700 | not detected | \<40 copies/g | | AUG 10 | 15 | 20 | not detected | \<40 copies/g | | SEP 10 | 14 | 170 | not detected | \<40 copies/g | | OCT 10 | 6 | 130 | not detected | \<40 copies/g | | NOV 10 | 8 | 1300 | not detected | \<40 copies/g | | DEC 10 | 2 | 700 | not detected | \<40 copies/g | | JAN 11 | 6 | 330 | positive | \<100 copies/g | | FEB 11 | 5 | 210 | positive | \<100 copies/g | | MAR 11 | 6 | \<20 | not detected | \<40 copies/g | | APR 11 | 12 | 90 | positive | \<100 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 80 | not detected | \<40 copies/g | positive | \<100 copies/g | | JUN 09 | 13 | 2400 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 09 | 18 | 2400 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 09 | 14 | 5400 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 09 | 14 | 2400 | positive | \<100 copies/g | not detected | \<40 copies/g | | OCT 09 | 14 | 330 | not detected | \<40 copies/g | not detected | \<40 copies/g | | NOV 09 | 12 | 1300 | not detected | \<40 copies/g | not detected | \<40 copies/g | | DEC 09 | 9 | 170 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JAN 10 | 5 | 70 | not detected | \<40 copies/g | not detected | \<40 copies/g | | FEB 10 | 5 | \<20 | positive | 1123 copies/g | not detected | \<40 copies/g | | MAR 10 | 8 | \<20 | positive | \<100 copies/g | not detected | \<40 copies/g | | APR 10 | 5 | 220 | not detected | \<40 copies/g | not detected | \<40 copies/g | | MAY 10 | 5 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUN 10 | 7 | 130 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JUL 10 | 7 | 1300 | not detected | \<40 copies/g | not detected | \<40 copies/g | | AUG 10 | 10 | 9200 | not detected | \<40 copies/g | not detected | \<40 copies/g | | SEP 10 | 10 | 220 | not detected | \<40 copies/g | not detected | \<40 copies/g | | OCT 10 | 9 | 790 | not detected | \<40 copies/g | positive | \<100 copies/g | | NOV 10 | 6 | 130 | not detected | \<40 copies/g | not detected | \<40 copies/g | | DEC 10 | 6 | 20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | JAN 11 | 7 | 20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | FEB 11 | 8 | \<20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | MAR 11 | 7 | 50 | not detected | \<40 copies/g | not detected | \<40 copies/g | | APR 11 | 7 | 20 | not detected | \<40 copies/g | not detected | \<40 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |--------|-------------------|---------|--------------|---------------| | JUN 09 | 13 | 700 | not detected | \<40 copies/g | | JUL 09 | 13 | 70 | no result | no result | | AUG 09 | 15 | \<20 | positive | \<100 copies/g | | SEP 09 | 14 | 490 | no result | no result | | OCT 09 | 14 | \<20 | positive | \<100 copies/g | | NOV 09 | 11 | 1300 | not detected | \<40 copies/g | | DEC 09 | 9 | 80 | positive | \<100 copies/g | | JAN 10 | 5 | \<20 | positive | \<100 copies/g | | FEB 10 | 5 | 80 | not detected | \<40 copies/g | | MAR 10 | 8 | 50 | not detected | \<40 copies/g | | APR 10 | 5 | \<20 | not detected | \<40 copies/g | | MAY 10 | 5 | 130 | not detected | \<40 copies/g | | JUN 10 | 7 | 80 | not detected | \<40 copies/g | | JUL 10 | 6 | 790 | not detected | \<40 copies/g | | AUG 10 | 10 | 130 | not detected | \<40 copies/g | | SEP 10 | 10 | 20 | not detected | \<40 copies/g | | OCT 10 | 8 | \<20 | positive | \<100 copies/g | | NOV 10 | 7 | 20 | not detected | \<40 copies/g | | DEC 10 | 6 | 330 | not detected | \<40 copies/g | | JAN 11 | 6 | 130 | not detected | \<40 copies/g | | FEB 11 | 8 | 20 | positive | \<100 copies/g | | MAR 11 | 7 | 20 | not detected | \<40 copies/g | | APR 11 | 7 | \<20 | not detected | \<40 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 10 | positive| \<100 copies/g| not detected | | JUN 09 | 14 | not detected| \<40 copies/g| not detected | | JUL 09 | 16 | positive| \<100 copies/g| not detected | | AUG 09 | 15 | positive| \<100 copies/g| positive | | SEP 09 | 13 | positive| \<100 copies/g| not detected | | OCT 09 | 12 | positive| \<100 copies/g| not detected | | JAN 10 | 5 | positive| \<100 copies/g| positive | | FEB 10 | 7 | \<20 | positive | 563 copies/g | | MAR 10 | 5 | positive| \<100 copies/g| positive | | APR 10 | 7 | 20 | positive | 534 copies/g | | MAY 10 | 11 | 20 | not detected | \<40 copies/g | | JUN 10 | 12 | 50 | positive | \<100 copies/g | | JUL 10 | 14 | 1300 | not detected | \<40 copies/g | | AUG 10 | 14 | 2400 | positive | \<100 copies/g | | SEP 10 | 13 | 170 | not detected | \<40 copies/g | | OCT 10 | 10 | 130 | not detected | \<40 copies/g | | NOV 10 | 8 | 790 | positive | \<100 copies/g | | JAN 11 | 7 | 50 | positive | 297 copies/g | | FEB 11 | 5 | 230 | positive | 160 copies/g | | MAR 11 | 6 | 50 | positive | 252 copies/g | | APR 11 | 10 | 170 | positive | \<100 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 10 | 5400 | not detected | \<40 copies/g | | | | | not detected | \<40 copies/g | | JUN 09 | 14 | 330 | positive | \<100 copies/g | | | | | not detected | \<40 copies/g | | JUL 09 | 15 | 490 | not detected | \<40 copies/g | | | | | not detected | \<40 copies/g | | AUG 09 | 14 | 50 | positive | \<100 copies/g | | | | | not detected | \<40 copies/g | | SEP 09 | 15 | 230 | positive | \<100 copies/g | | | | | positive | \<100 copies/g | | OCT 09 | 13 | 330 | not detected | \<40 copies/g | | | | | positive | \<100 copies/g | | NOV 09 | 12 | 330 | not detected | \<40 copies/g | | | | | not detected | \<40 copies/g | | DEC 09 | 10 | \<20 | not detected | \<40 copies/g | | | | | not detected | \<40 copies/g | | JAN 10 | 7 | 490 | no result | no result | | FEB 10 | 6.5 | 330 | positive | \<100 copies/g | | | | | positive | 235 copies/g | | MAR 10 | 7 | 230 | not detected | \<40 copies/g | | | | | positive | 100-500 copies/g | | APR 10 | 10 | \<20 | not detected | \<40 copies/g | | | | | positive | 100-500 copies/g | | MAY 10 | 10 | \<20 | positive | \<100 copies/g | | | | | not detected | \<40 copies/g | | JUN 10 | 11 | 20 | positive | \<100 copies/g | | | | | not detected | \<40 copies/g | | JUL 10 | 14 | 9200 | not detected | \<40 copies/g | | | | | not detected | \<40 copies/g | | AUG 10 | 15 | 170 | positive | \<100 copies/g | | | | | not detected | \<40 copies/g | | SEP 10 | 16 | 700 | positive | \<100 copies/g | | | | | not detected | \<40 copies/g | | OCT 10 | 13 | 330 | not detected | \<40 copies/g | | | | | not detected | \<40 copies/g | | NOV 10 | 10 | 20 | not detected | \<40 copies/g | | | | | positive | 1462 copies/g | | DEC 10 | 8 | 50 | not detected | \<40 copies/g | | | | | positive | 249 copies/g | | JAN 11 | 6 | 230 | positive | \<100 copies/g | | | | | positive | 1148 copies/g | | FEB 11 | 7 | \<20 | positive | \<100 copies/g | | | | | positive | 106 copies/g | | MAR 11 | 9 | \<20 | not detected | \<40 copies/g | | | | | not detected | \<40 copies/g | | APR 11 | 11 | 130 | not detected | \<40 copies/g | | | | | not detected | \<40 copies/g | | Month | Water temperature | E. coli | Norovirus GI | Norovirus GII | |---------|-------------------|---------|--------------|---------------| | MAY 09 | 10 | 80 | not detected | \<40 copies/g | | | | | | positive | | JUN 09 | 14 | 790 | not detected | \<40 copies/g | | | | | | not detected | | JUL 09 | 15 | 330 | not detected | \<40 copies/g | | | | | | not detected | | AUG 09 | 16 | 1300 | positive | \<100 copies/g | | | | | | not detected | | SEP 09 | 14 | 50 | not detected | \<40 copies/g | | | | | | not detected | | OCT 09 | 12 | 790 | not detected | \<40 copies/g | | | | | | not detected | | NOV 09 | 11 | 790 | positive | \<100 copies/g | | | | | | positive | | DEC 09 | 8 | 50 | positive | \<100 copies/g | | | | | | positive | | JAN 10 | 7 | 50 | positive | \<100 copies/g | | | | | | positive | | FEB 10 | 6 | \<20 | positive | \<100 copies/g | | | | | | positive | | MAR 10 | 6 | 20 | positive | \<100 copies/g | | | | | | not detected | | APR 10 | 9 | 80 | not detected | \<40 copies/g | | | | | | positive | | MAY 10 | 10 | \<20 | not detected | \<40 copies/g | | | | | | not detected | | JUN 10 | 14 | \<20 | not detected | \<40 copies/g | | | | | | not detected | | JUL 10 | 14 | 2200 | not detected | \<40 copies/g | | | | | | not detected | | AUG 10 | 15 | 790 | not detected | \<40 copies/g | | | | | | not detected | | SEP 10 | 15 | \<20 | not detected | \<40 copies/g | | | | | | not detected | | OCT 10 | 13 | 220 | not detected | \<40 copies/g | | | | | | not detected | | NOV 10 | 9 | 70 | positive | \<100 copies/g | | | | | | not detected | | DEC 10 | 7 | 20 | positive | \<100 copies/g | | | | | | positive | | JAN 11 | 6 | 50 | not detected | \<40 copies/g | | | | | | positive | | FEB 11 | 5 | 20 | positive | \<100 copies/g | | | | | | positive | | MAR 11 | 9 | \<20 | positive | \<100 copies/g | | | | | | not detected | | APR 11 | 9 | 490 | positive | 533 copies/g | | | | | | not detected | Appendix 3: Statistical analysis

Page 6: Oyster species comparison Two-way ANOVA: norovirus versus species, contamination

| Source | DF | SS | MS | F | P | |--------------|----|--------|--------|--------|-------| | species | 1 | 0.0614 | 0.06138| 1.46 | 0.232 | | contamination| 2 | 18.4199| 9.20993| 219.40 | 0.000 | | Interaction | 2 | 0.1257 | 0.06287| 1.50 | 0.233 | | Error | 54 | 2.2668 | 0.04198| | | | Total | 59 | 20.8738| | | |

S = 0.2049 R-Sq = 89.14% R-Sq(adj) = 88.13%

Individual 95% CIs For Mean Based on Pooled StDev

| species | Mean | 95% CI | |---------|------|-----------------| | C.gigas | 4.60887 | (4.560, 4.620) | | O.edulis| 4.67284 | (4.620, 4.680) |

Individual 95% CIs For Mean Based on Pooled StDev

| contamination | Mean | 95% CI | |---------------|------|-----------------| | high | 5.23487 | (4.00, 5.20) | | medium | 4.78641 | (4.40, 4.80) | | low | 3.90129 | (3.90, 4.00) |

Page 10: GI vs GII Tabulated statistics: genogroup, result

Using frequencies in C3

Rows: genogroup Columns: result

| | - | + | All | |-----|---|---|-----| | GI | 266 | 578 | 844 | | GII | 377 | 467 | 844 | | All | 643 | 1045 | 1688 |

Cell Contents: Count

Fisher's exact test: P-Value = 0.0000000 Page 10: Summer vs winter prevalence Tabulated statistics: season, norovirus

Using frequencies in C3

Rows: season Columns: norovirus

- - All summer 159 264 423 winter 42 379 421 All 201 643 844

Cell Contents: Count

Fisher's exact test: P-Value = 0.000000

Page 11: GI vs GII Wilcoxon Signed Rank Test: Diff

Test of median = 0.000000 versus median not = 0.000000

| N for Wilcoxon | Estimated | |----------------|-----------| | N Test Statistic P Median | | Diff 643 521 44434.0 0.000 -39.50 |

Wilcoxon Signed Rank Test: Diff; \<LOD = 0, \<LOQ = 1

Test of median = 0.000000 versus median not = 0.000000

| N for Wilcoxon | Estimated | |----------------|-----------| | N Test Statistic P Median | | Diff 643 521 43352.0 0.000 -60.55 |

Wilcoxon Signed Rank Test: Diff; \<LOD = 40, \<LOQ = 100

Test of median = 0.000000 versus median not = 0.000000

| N for Wilcoxon | Estimated | |----------------|-----------| | N Test Statistic P Median | | Diff 643 521 48094.0 0.000 -30.00 |

Page 11: Spearman's rank of GI vs GII Correlations: GIrank, GIIrank

Pearson correlation of GIrank and GIIrank = 0.622 P-Value = 0.000 Page 13: Yr1 vs Yr2 prevalence Tabulated statistics: year, norovirus

Using frequencies in C3

Rows: year Columns: norovirus

- - All yr1 91 337 428 yr2 110 306 416 All 201 643 844

Cell Contents: Count

Fisher's exact test: P-Value = 0.0895321

Page 13: Yr1 vs Yr2 Kruskal-Wallis Test: norovirus versus year

Kruskal-Wallis Test on norovirus

| year | N | Median | Ave Rank | Z | |------------|----|--------|----------|-----| | yr1 | 428| 100.0 | 417.1 | -0.65| | yr2 | 416| 100.0 | 428.1 | 0.65 | | Overall | 844| | 422.5 | |

H = 0.43 DF = 1 P = 0.513 H = 0.44 DF = 1 P = 0.506 (adjusted for ties)

Page 14: Between regions comparison Kruskal-Wallis Test: norovirus versus region

Kruskal-Wallis Test on norovirus

| region | N | Median | Ave Rank | Z | |---------------------------------------------|----|--------|----------|-----| | England - East Coast | 12 | 226.30 | 22.8 | 1.00| | England - South Coast | 13 | 194.60 | 23.9 | 1.52| | England - West Coast, Wales and NI | 8 | 118.74 | 18.5 | -0.42| | Scotland | 6 | 81.92 | 8.0 | -2.80| | Overall | 39 | | 20.0 | |

H = 9.02 DF = 3 P = 0.029 Page14: Between regions comparison – post hoc analysis Kruskal-Wallis: All Pairwise Comparisons

Comparisons: 6 Family Alpha: 0.2 Bonferroni Individual Alpha: 0.033 Bonferroni Z-value (2-sided): 2.128

Standardized Absolute Mean Rank Differences | | / Stdev

Rows: Group i = 1,...,n Columns: Group j = 1,...,n

1. Table of Z-values

| | * | * | * | England - East Coast | 0.00000 | | | | England - South Coast | 0.25701 | 0.00000 | * | * | England - West Coast, Wales & NI | 0.81665 | 1.05848 | 0.00000 | * | Scotland | 2.58732 | 2.82961 | 1.70520 | 0

2. Table of P-values

| | * | * | * | England - East Coast | 1.00000 | | | | England - South Coast | 0.79717 | 1.00000 | * | * | England - West Coast, Wales & NI | 0.41413 | 0.28984 | 1.00000 | * | Scotland | 0.00967 | 0.00466 | 0.08816 | 1

Sign Confidence Intervals controlled at a family error rate of 0.2

Desired Confidence: 86.761

Sign confidence interval for median

| | Achieved Confidence | Confidence Interval | Position | | | Lower | Upper | | | norovirus_England - East Coast | 12 | 226.3 | 0.8540 | 90.0 | 393.3 | 4 | | norovirus_England - South Coast | 13 | 194.6 | 0.7332 | 173.9 | 200.5 | 5 | | norovirus_England - West Coast, Wales & NI | 8 | 118.7 | 0.7109 | 112.8 | 175.3 | 3 | | norovirus_Scotland | 6 | 81.9 | 0.7813 | 52.0 | 95.7 | 2 |

Kruskal-Wallis: Conclusions

The following groups showed significant differences:

| Groups | Z vs. Critical value | P-value | | England - South Coast vs. Scotland | 2.82961 >= 2.128 | 0.0047 | | England - East Coast vs. Scotland | 2.58732 >= 2.128 | 0.0097 | Page 15: Between species comparison Kruskal-Wallis Test: norovirus versus species

Kruskal-Wallis Test on norovirus

| species | N | Median | Ave Rank | Z | |------------|----|--------|----------|------| | C.gigas | 28 | 164.9 | 19.9 | -0.12| | O.edulis | 11 | 173.9 | 20.4 | 0.12 | | Overall | 39 | | 20.0 | |

H = 0.02 DF = 1 P = 0.901

Page 16: Between classifications comparison Kruskal-Wallis Test: norovirus versus classification

Kruskal-Wallis Test on norovirus

| classification | N | Median | Ave Rank | Z | |----------------|----|--------|----------|------| | A | 6 | 81.92 | 8.7 | -2.65| | B | 31 | 179.55 | 21.5 | 1.67 | | C | 2 | 1208.33| 30.0 | 1.27 | | Overall | 39 | | 20.0 | |

H = 8.04 DF = 2 P = 0.018

- NOTE * One or more small samples

Page 16: Between classifications comparison – post hoc analysis Kruskal-Wallis: All Pairwise Comparisons

Comparisons: 3 Family Alpha: 0.2 Bonferroni Individual Alpha: 0.067 Bonferroni Z-value (2-sided): 1.834

Standardized Absolute Mean Rank Differences |Rbar(i)-Rbar(j)| / Stdev

Rows: Group i = 1,...,n Columns: Group j = 1,...,n

1. Table of Z-values

| | A | B | C | |-----|-----|-----|-----| | A | 0.00000 | * | * | | B | 2.53313 | 0.00000 | * | | C | 2.29157 | 1.01603 | 0 |

2. Table of P-values

| | A | B | C | |-----|-----|-----|-----| | A | 1.00000 | * | * | | B | 0.01130 | 1.00000 | * | | C | 0.02193 | 0.30961 | 1 | Sign Confidence Intervals controlled at a family error rate of 0.2

Desired Confidence: 80.529

Sign confidence interval for median

| norovirus_1_A | N | Median | Confidence Achieved | Lower | Upper | Position | |---------------|----|--------|---------------------|-------|-------|----------| | norovirus_1_B | 31 | 179.5 | 0.7190 | 138.3 | 197.1 | 13 | | norovirus_1_C | 2 | 1208 | 0.5000 | 174 | 2243 | 1 |

The highest attainable confidence has been achieved.

Kruskal-Wallis: Conclusions

The following groups showed significant differences:

| Groups | Z vs. Critical value | P-value | |-----------------|----------------------|---------| | A vs. B | 2.53313 >= 1.834 | 0.0113 | | A vs. C | 2.29157 >= 1.834 | 0.0219 |

Page 17: Spearman’s rank norovirus vs. E. coli sample-by-sample Correlations: ranknorovirus, rankE.coli

Pearson correlation of ranknorovirus and rankE.coli = 0.195 P-Value = 0.000

Page 18: Spearman’s rank norovirus vs. E. coli site-by-site Correlations: ranknorovirus, rankE.coli

Pearson correlation of ranknorovirus and rankE.coli = 0.453 P-Value = 0.004

Page 18: Spearman’s rank norovirus vs. E. coli site-by-site, winter Correlations: ranknorovirus, rankE.coli

Pearson correlation of ranknorovirus and rankE.coli = 0.676 P-Value = 0.000

Page 18: Spearman’s rank norovirus in oysters vs. HPA lab reports Correlations: rankoyster, rankHPA

Pearson correlation of rankoyster and rankHPA = 0.761 P-Value = 0.000 Page 20: Spearman's rank norovirus in oysters vs. UK average air temperatures Correlations: rankoyster, ranktemp

Pearson correlation of rankoyster and ranktemp = -0.877 P-Value = 0.000

Page 20: Spearman's rank norovirus in oysters vs. Water temperatures (individual sites) Site 1 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.797 P-Value = 0.000

Site 2 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.783 P-Value = 0.000

Site 3 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.528 P-Value = 0.014

Site 4 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.879 P-Value = 0.000

Site 7 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.766 P-Value = 0.001

Site 8 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.676 P-Value = 0.001

Site 9 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.596 P-Value = 0.003 Site 10 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.603 P-Value = 0.002

Site 11 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.865 P-Value = 0.000

Site 12 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.755 P-Value = 0.000

Site 13 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.747 P-Value = 0.003

Site 14 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.433 P-Value = 0.160

Site 15 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.737 P-Value = 0.000

Site 16 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.573 P-Value = 0.005

Site 17 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.870 P-Value = 0.000 Site 18 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.587 P-Value = 0.027

Site 20 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.737 P-Value = 0.004

Site 21 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.359 P-Value = 0.208

Site 22 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.862 P-Value = 0.000

Site 23 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.783 P-Value = 0.001

Site 24 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = 0.002 P-Value = 0.992

Site 25 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.704 P-Value = 0.001

Site 26 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.690 P-Value = 0.000 Site 27 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.549 P-Value = 0.008

Site 28 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.407 P-Value = 0.060

Site 29 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.618 P-Value = 0.003

Site 30 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.330 P-Value = 0.124

Site 31 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.615 P-Value = 0.005

Site 32 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.242 P-Value = 0.333

Site 33 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.832 P-Value = 0.000

Site 34 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.373 P-Value = 0.080 Site 35 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = 0.013 P-Value = 0.954

Site 36 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.142 P-Value = 0.539

Site 37 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.754 P-Value = 0.000

Site 38 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.460 P-Value = 0.027

Site 39 Correlations: ranktemp, ranknoro

Pearson correlation of ranktemp and ranknoro = -0.746 P-Value = 0.000

Page 20: Spearman's rank norovirus vs. risk score Correlations: rankrisk, ranknorovirus

Pearson correlation of rankrisk and ranknorovirus = 0.546 P-Value = 0.001

Page 21: Spearman's rank norovirus vs. sampling risk score Correlations: ranksampling risk score, ranknorovirus

Pearson correlation of ranksampling risk score and ranknorovirus = 0.645 P-Value = 0.000